Environmentally regulated genes of Streptococcus suis

ABSTRACT

The invention relates to the field of the diagnosis of and vaccination against Streptococcal infections, and to the detection of virulence markers of Streptococci. The invention discloses a method for modulating virulence of a Streptococcus comprising modifying a genomic fragment of the Streptococcus, wherein the genomic fragment comprises at least a functional part of a fragment identifiable by hybridization in  Streptococcus suis  to a nucleic acid or fragment thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of PCT/NL02/00073, filed Jan.31, 2002, designating the United States of America, corresponding to PCTInternational Publication WO 02/061070 (published in English on Aug. 8,2002), the contents of which are incorporated herein in its entirety.

TECHNICAL FIELD

[0002] The invention relates to the field of the diagnosis of andvaccination against Streptococcal infections, and to the detection ofvirulence markers of Streptococci.

BACKGROUND

[0003] Streptococcus species, of which there are a large variety of thatcause infections in domestic animals and man, are often groupedaccording to Lancefield's groups. Typing according to Lancefield occurson the basis of serological determinants or antigens that are, amongothers, present in the capsule of the bacterium and only allows for anapproximate determination. Often, bacteria from a different group showcross-reactivity with each other, while other Streptococci cannot beassigned a group-determinant at all. Within groups, furtherdifferentiation is often possible on the basis of serotyping. Theseserotypes further contribute to the large antigenic variability ofStreptococci, a fact that creates an array of difficulties withindiagnosis of and vaccination against Streptococcal infections.

[0004] Lancefield group A Streptococcus (GAS, Streptococcus pyogenes)are common with children and cause nasopharyngeal infections andcomplications thereof. Animals, such as cattle, are susceptible to GAS,wherein mastitis is often found associated with the cattle.

[0005] Lancefield group B Streptococcus (GBS) are most often seen withcattle and cause mastitis. However, human infants are susceptible aswell, often with fatal consequences. Group B Streptococci (GBS)constitute a major cause of bacterial sepsis and meningitis among humanneonates born in the United States and Western Europe and are emergingas significant neonatal pathogens in developing countries.

[0006] Lancefield group C infections, such as those with S. equi, S.zooepidemicus, S. dysgalactiae, and others are mainly seen associatedwith horses, cattle and pigs, but can also cross the species barrier tohumans.

[0007] Lancefield group D (S. bovis) infections are found with allmammals and some birds, sometimes resulting in endocarditis orsepticemia.

[0008] Lancefield groups E, G, L, P, U and V (S. porcinus, S. canis, S.dysgalactiae) are found with various hosts and cause neonatalinfections, nasopharyngeal infections or mastitis.

[0009] Within Lancefield groups R, S, and T (and with ungrouped types),S. suis is found and is an important cause of meningitis, septicemia,arthritis and sudden death in young pigs. Incidentally, S. suis can alsocause meningitis in man.

[0010] Ungrouped Streptococcus species, such as S. mutans, causescarries with humans. S. uberis causes mastitis with cattle, S. pneumoniacauses major infections in humans, and Enterococcus faecilalis and E.faecium further contribute to the large group of Streptococci.Streptococcus pneumoniae (the pneumococcus) is a human pathogen causinginvasive diseases, such as pneumonia, bacteremia, and meningitis.

[0011] Little is known about the pathogenesis of the disease caused byStreptococci. Various cellular components, such as muramidase-releasedprotein (MRP), extracellular factor (EF) and cell-membrane-associatedproteins including fimbriae, hemagglutinins, and hemolysin have beensuggested as virulence factors. However, the precise role of theseprotein components in the pathogenesis of the disease remains unclear.It is known and generally accepted that the polysaccharidic capsule ofvarious Streptococci and other gram-positive bacteria play an importantrole in pathogenesis. The capsule enables these microorganisms to resistphagocytosis and is, therefore, regarded as an important virulencefactor or marker.

[0012] In particular, Streptococcus suis is an important cause ofmeningitis, septicemia, arthritis and sudden death in young pigs. It canalso cause meningitis in man. Attempts to control the disease arehampered by the lack of sufficient knowledge about the pathogenesis ofthe disease and the lack of effective vaccines and sensitive diagnosticmethods.

[0013] So far, 35 serotypes of S. suis are described. Virulence of S.suis can differ within and among serotypes. Worldwide, S. suis serotype2 is the most frequently isolated serotype. Within S. suis serotype 2,pathogenic, weak-pathogenic and non-pathogenic strains can be found. Thepathogenic strains cause severe clinical signs of disease in pigs andlarge numbers of bacteria can be re-isolated from the central nervoussystem (CNS) and the joints after experimental infection. Theweak-pathogenic strains cause only mild clinical signs of disease andinfrequently are bacteria re-isolated from the CNS and the joints afterexperimental infection. The non-pathogenic strains are completelyavirulent in young pigs after experimental infection.

[0014] The 136-kDa muramidase-related protein (MRP) and the 110-kDaextracellular factor (EF) are generally considered as importantvirulence markers for S. suis serotype 2 strains isolated in Europe andthe United States. However, differences in virulence between pathogenic,weak-pathogenic and non-pathogenic strains cannot exclusively beexplained by differences in their MRP and EF expression patterns. Inaddition, it is known that the capsule of Streptococcus suis serotype 2is an important virulence factor. However, since pathogenic,weak-pathogenic and non-pathogenic strains seem to be fully encapsulatedafter growth in vitro and in vivo, it is not likely that the level ofencapsulation of these fully encapsulated strains is associated withtheir difference in virulence.

SUMMARY OF THE INVENTION

[0015] The invention discloses a method for modulating virulence of aStreptococcus comprising modifying a genomic fragment of Streptococcus.The genomic fragment comprises at least a functional part of a fragmentidentifiable by hybridization in Streptococcus suis to a nucleic acid orfragment thereof as shown in SEQ ID NOS: 8 through 45 and obtaining aclone including the modified genomic fragment. In one exemplaryembodiment, the genomic fragment comprises at least a functional part ofa gene, the expression of which can be environmentally regulated in S.suis by iron-restricted conditions. In another exemplary embodiment, thegenomic fragment comprises at least a functional part of a gene which isexpressed in a wild-type S. suis infected pig (in vivo). In a furtherexemplary embodiment, selection under iron-restricted conditions iscombined with selection in vivo. In one embodiment, the gene encodes afibronectin/fibrinogen-binding protein. The method disclosed herein isuseful for modulating virulence of Streptococcus suis and comprisesfunctionally deleting the expression of at least the functional part ofthe gene by Streptococcus.

[0016] The phrase “functionally deleting” as used herein refers to anytechnique known in the art (such as allowing for a deletion, insertion,mutation or the occurrence of a frame-shift in the open-reading frame ofthe respective gene) that is instrumental in hampering or inhibiting theexpression of a gene-product (be it mRNA and/or protein) of the gene.Thus, the invention discloses a clone of a Streptococcus obtained orobtainable by a method according to the invention.

[0017] To get insight in the differences between pathogenic,weak-pathogenic and nonpathogenic strains or clones that are determinedby their difference in virulence, the invention describes theidentification of environmentally regulated genes of Streptococcus suisby iron-restricted conditions and by experimental infection of piglets.Eighteen unique iron-restricted induced (iri) genes and 22 unique invivo selected (ivs) genes of S. suis were found. None of the ivs geneswas exclusively expressed in vivo. Four iri genes were substantiallyidentical to four ivs genes selected in piglets. Two ivs genes weresimilar to genes for putative virulence factors. One of these ivs geneswas substantially identical to the epf gene of virulent S. suis serotype2 strains and the other ivs gene showed homology to a gene encoding afibronectin-binding protein of Streptococcus gordonii.

[0018] In yet another embodiment, the invention discloses a study of thecharacteristics of fibronectin- and fibrinogen-binding protein ofStreptococcus suis (FBPS) and its gene as identified herein. The abilityto bind fibronectin, either in fluid phase or immobilized onto asurface, is a property of S. suis and is one of the mechanisms S. suisuses for attachment to and invasion of host cells. Therefore, FBPS is animportant virulence factor. The gene encoding FBPS was identified usingan in vivo selection system in pigs as described herein, thus, showingan important role of the protein in vivo. This finding was supported bythe observation that isogenic FBPS mutants, herein also disclosed, of S.suis are attenuated in pigs. Surprisingly, FBPS bound to fibronectin, aswell as to fibrinogen, but did not show the structural characteristicsof the fibronectin-binding proteins most commonly described and explainswhy FBPS has not been found earlier. Most fibronectin-binding proteinsdescribed to date are large cell surface proteins with predicted sizesof 60-100 kDa and have similar structural organizations. The proteinscontain an N-terminal signal sequence as well as the cell wall signalingsequence (LPXTGE) (SEQ ID NO: 1). The Fn-binding sites include 30-42amino acid long motifs, repeated 3-4 times. In particular, the firstfibronectin- and fibrinogen-binding protein of S. suis is disclosedherein. The gene encoding FBPS was cloned and sequenced and FBPS waspurified. Binding of FBPS to human fn and fgn was shown. FBPS was shownto be involved in the colonization of the organs specific for an S. suisinfection in piglets, but not in the colonization of S. suis on thetonsils of piglets.

[0019] Many Streptococci and Staphylococci have several differentfibronectin- and/or fibrinogen-binding proteins, most of which are verylarge, about 130 kDa. Until now, S. pyogenes was the only organism tohave a large, as well as a smaller (54 kDa), FnBP. The existence of morethan one FnBP explains why in some organisms, isogenic mutants defectivein only one of the FnBPs can still bind to fn and/or fgn can be furtherattenuated in vivo in relation to fibronectin binding.

[0020] The role of FBPS in the pathogenesis of S. suis was studied in anexperimental infection model in piglets. Since we were unable todetermine a LD₅₀ values for the mutant clones because no lethal dosecould be established using normally used numbers of bacteria, it wasdecided to compare the virulence of the isogenic FBPS clone to thewild-type S. suis in a competitive infection assay in piglets. This kindof co-colonization experiment has been successfully applied to determinethe virulence of mutants of Actinobacillus pleuropneumoniae in piglets.The data showed that the mutant clone was capable of colonizing thetonsil as efficiently as the wild-type. This strongly indicates thatFBPS is not involved in the colonization of the tonsil. The data alsoindicated that FBPS does play a role in the colonization of specificorgans, since in the competition assay, joints and the CNS were moreefficiently colonized by wild-type than by mutant bacteria.

[0021] In addition, higher numbers of wild-type bacteria werere-isolated from the specific organs compared to the numbers of mutantbacteria, indicating that the mutant clone is attenuated in vivo.Although the number of pigs used for this experiment was low, the dataindicates that the FBPS mutant is less virulent than the wild-typestrain. It was demonstrated that FBPS reacted with a convalescent serumof a pig that survived an S. suis infection. Therefore, FBPS isimmunogenic in pigs and this finding demonstrates that FBPS of S. suisis expressed under in vivo conditions.

[0022] It is also shown that the fbps gene was present in all knownserotypes of S. suis (except for two), as well as in all threephenotypes of serotype 2. This suggests that the fbps gene is presentamong most serotypes. However, the expression of FBPS in all serotypesand phenotypes was not studied. Therefore, it is possible that althoughall strains, except for serotypes 32 and 34, possess the fbps gene, notall strains express FBPS. Based on the facts that FBPS is immunogenic inpigs and that the fbps gene is present in all prevailing S. suisserotypes, (except for two), FBPS is an attractive candidate for across-protective vaccine against essentially all serotypes. In oneembodiment, the mutant strain 10ΔFBPS may be used in the vaccine, whichmutant is possibly further attenuated by deleting one or more virulencefactors as described herein. In another embodiment, this vaccine isbased on purified FBPS protein or an antigenic part thereof with asuitable adjuvant.

[0023] To further validate a method for identifying a virulence factor,the role of the fibronectin-/fibrinogen-binding protein (FBPS) in thepathogenesis of S. suis serotype 2 was investigated in piglets asdescribed herein. The complete gene encoding FBPS from S. suis serotype2 was cloned in E. coli and sequenced. The occurrence of the gene invarious serotypes was analyzed by hybridization studies. The FBPSprotein was expressed in E. coli, purified and binding to humanfibronectin and fibrinogen was demonstrated. The induction of antibodiesin piglets was studied upon infection. An isogenic mutant unable toproduce FBPS was constructed and the virulence of the wild-type andmutant strains was compared in a competitive infection model in youngpiglets. Organ cultures showed that FBPS was not required forcolonization of the tonsils, but that FBPS played a role in thecolonization of the specific organs involved in an S. suis infection.Therefore, the FBPS mutant was considered as an attenuated mutant whichis useful in a vaccine. Alternatively, a vaccine is used that mainlyincludes the FBPS protein or at least of an antigenic part thereof, suchthat an FBPS-specific antibody or T-cell response in pigs is developedafter vaccination with the FBPS or antigenic part thereof.

[0024] Two additional ivs genes showed homology to environmentallyregulated genes previously identified by using an in vivo expressiontechnology (IVET) selection in other bacterial species. One of theseshowed similarity to the agrA gene of Staphylococcus aureus, a key locusinvolved in the regulation of numerous virulence proteins.

[0025] Thus, the invention also discloses a method for assayingvirulence of a Streptococcus comprising assaying a genomic fragment ofStreptococcus, wherein the genomic fragment comprises at least afunctional part of a fragment identifiable by hybridization inStreptococcus suis to a nucleic acid or fragment thereof as describedherein.

[0026] The invention also discloses a vector comprising a nucleic acidaccording to the invention and a host cell comprising a nucleic acid ora vector according to the invention. Such a host cell comprises aneasily modifiable organism such as E. coli. However, other host cells,such as a recombinant Streptococcus comprising a vector. or nucleic acidaccording to the invention are also disclosed herein.

[0027] The invention additionally discloses a vaccine comprising anucleic acid, a vector or a host cell according to the invention, anduse of such a vaccine in the prevention and/or treatment ofStreptococcal infections.

[0028] Also disclosed is a protein or fragment thereof encoded by anucleic acid according to the invention, such as a protein encoded by anucleic acid or fragment thereof disclosed herein or functional, i.e.,antigenic fragment thereof. The invention also discloses an antibodydirected against a protein or fragment thereof according to theinvention and an antigen reactive with such an antibody, for examplecomprising a protein or fragment. Such a protein or fragment thereofneed not be obtained by recombinant means. Synthesis of peptides,according to their amino acid sequence, is as well equally possible.Such antigens and antibodies as described herein can be used in adiagnostic test comprising an antibody according to the invention, orwithin a vaccine or diagnostic test comprising an antigen according tothe invention. Such vaccines and diagnostic tests can be used in thefield of the diagnosis of and vaccination against Streptococcalinfections and for the detection of virulence markers of Streptococci.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 is a schematic presentation of the procedure used to clonethe fbps gene of S. suis serotype 2 and the construction of aninsertional knock-out mutant in S. suis serotype 2. A 5 kb EcoRIfragment was cloned in pGEM7Zf(+), yielding pFBPS7-46. In pFBPS7-47, the382 bp SalI-SalI fragment of pFBPS7-46 was replaced by 1.2 kbspectinomycin-resistance gene, after the vector was made blunt to obtainan insertional knock-out of fbps. Ivs-31: in vivo selected gene 31.

[0030]FIG. 2 shows purity and immunogenicity of FBPS purified undernative conditions. SDS-PAGE analysis with SYPRO orange, a non-specificprotein-staining (panel A) and Western blot analysis with a monoclonalantibody against the 6×HIS tag (panel B) of 4:1 of E. coli M15(pQE-3O-pREP4-FBPS) lysate (lanes 1) and 165 ng of purified FBPS (lanes2). Convalescent serum raised against S. suis strain 10 was used to testimmunogenicity of FPBS present in 4:1 of E. coli M15 (pQE-30-pREP4-FBPS)lysate and 0.5 μg of purified FBPS (Panel C, lanes 1 and 2). Arrowhead,64 kDa FPBS; Mw, molecular weight marker.

[0031]FIG. 3 depicts the binding studies with purified FBPS. Panels Aand B were probed with 5 μg/ml of fn (A) or fgn (B). Lanes 1 contain 500ng of purified FBPS, lanes 2 contain 500 ng of BSA. Panels C and D,lanes 3 and 4 contain 500 ng of purified FBPS. Lanes 3 were probed with20 μg/ml of fn (C) or fgn (D), lanes 4 were incubated with conjugatewithout fn or fgn. Panels E and F were probed with 20 μg/ml of fn (E) orfgn (F). Lanes 5 contain 1.8 μg of purified FBPS digested withenterokinase, lanes 6 contain 500 ng of purified FBPS. The closedarrowhead indicates 64 kDa FBPS; the open arrowhead indicatesapproximately 55 kDa FBPS without 6×HIS.

[0032]FIG. 4 shows the distribution of fbps among various S. suisserotypes. 1 μg of chromosomal DNA was spotted onto nitrocellulosemembrane and hybridized with a ³²P-labelled fbps probe. Serotypes werespotted as indicated. S10: S. suis serotype 2, MRP⁺EF⁺; T15: S. suisserotype 2 MRP⁻EF⁻; S17:S. suis serotype 2 MRP⁺EF*.

[0033]FIG. 5 illustrates the efficiency of colonization of wild-type andmutant bacteria on various organs of infected pigs. Panel A depictscolonization of the wild-type strain 10 and the mutant strain 10ΔFBPS ofthe tonsils. A closed diamond symbol is tonsil pig no. 4664; ▪ tonsilpig no. 4665; ▴ tonsil pig no 4666; • tonsil pig no. 4668. Panel Bdepicts colonization of the specific organs. Open and closed diamondsymbols are pus from joints pig no. 4664; ▴ pus from joint pig no. 4666;• CNS pig no. 4668. Each dot represents the numbers of wild-type ormutant bacteria isolated from one particular organ, from one piglet.

DETAILED DESCRIPTION

[0034]Streptococcus suis is an important cause of meningitis,septicemia, arthritis and sudden death in young pigs (Clifton-Haclley,1983; Vecht et al., 1985). It can also cause meningitis in man (Arendsand Zanen, 1988). Attempts to control the disease are still hampered bythe lack of sufficient knowledge about the pathogenesis of the disease,the lack of effective vaccines and sensitive diagnostic methods. To meetthese shortages, it is necessary to identify the genes that are involvedin the pathogenic process. So far, only a limited number of S. suisgenes are known (Smith et al., 1992; Smith et al., 1993; Serhir et al.,1997; Segers et al., 1998; Smith et al., 1999; and accession nos.AF106927, Z95920 and A57222) and of these, only a few are putativelyinvolved in virulence (Smith et al., 1992; Smith et al., 1993; Jacobs etal., 1994; Gottschalk et al., 1995; Segers et al., 1998; Smith et al.,1999). Previously, putative virulence factors have been identified aftergrowth of the bacteria in standard laboratory media. However, it isknown that many important virulence factors are environmentallyregulated and are induced at specific stages of the infection process(Mahan et al., 1993).

[0035] Recently, several approaches have been reported that allow theidentification of genes that are specifically expressed in the host.Examples are signature-tagged mutagenesis (STM) and in vivo expressiontechnology (IVET; Mahan et al., 1993; Carnilli and Mekalanos, 1995;Hensel et al., 1995; Mahan et al., 1995; Mei et al., 1997; Young andMiller, 1997; Chiang and Mekalanos, 1998; Coulter et al., 1998; Lowe etal., 1998; Polissi et al., 1998; Camacho et al., 1999; Darwin et al.,1999; Edeistein et al., 1999; Fuller et al., 1999; Zhao et al., 1999).In addition, important virulence proteins could also be identified bythe selection of genes specifically expressed under conditions mimickingin vivo conditions, for example by growth in iron-restricted conditions(Litwin and Calderwood, 1993; Martinez et al., 1990).

[0036] The present invention identifies virulence genes of S. suis byselecting environmentally regulated genes by experimental infections ofpiglets and by the use of iron-restricted conditions in vitro. For thispurpose, chromosomal DNA fragments of S. suis were cloned in a plasmidin front of a promoterless erythromycin-resistance gene. Subsequently,the library was used for the selection of bacteria in which erythromycinresistance was induced under iron-restricted conditions. In addition,erythromycin-resistant bacteria were selected after infection of pigletswith the library and treatment of the piglets with erythromycin. Pigswere used instead of mice for these experiments since it was recentlyshown that virulence of S. suis is different in these two animal species(Vecht et al., 1997). Using this approach, 18 uniqueiron-restriction-induced (iri) genes, as well as 22 unique in vivoselected (ivs) genes, were identified, several of which are putativelyinvolved in virulence (Smith et al., 1993; Smith et al., 1996).

[0037] Methods.

[0038] Bacterial strains and growth conditions. The bacterial strainsand plasmids used in this study are listed in Table 1. S. suis strainswere grown in Todd-Hewitt broth (Oxoid), and plated on Columbia agar(Oxoid) containing 6% (v/v) horse blood. For the selection of genesinduced in iron-limited conditions, S. suis cells. were plated on agarplates containing Todd-Hewitt medium, 5% (w/v) yeast extract and 75 μMdeferoxamine mesylate (Sigma). Control plates were supplemented with 38μM FeSO₄.7H₂O (Sigma). If required, antibiotics were added at thefollowing concentrations: 100 μg spectinomycin ml⁻¹and 1 μg erythromycinml⁻¹ . E. coli strains were grown in Luria broth (Miller, 1972) andplated on Luria broth containing 1.5% (w/v) agar. If required, 50 μgampicillin ml⁻¹ or 50 μg spectinomycin ml⁻¹ was added.

[0039] Construction of pIVS-E.

[0040] The IVS selection vector used in this study comprises aspectinomycin-resistance gene, a promoterless erythromycin-resistancegene and the origin of replication of the plasmid pWVO1 (Van der Vossenet al., 1987). To construct this pIVS-E, the spectinomycin-resistancegene was amplified from pKUN19-spc (Konings et al., 1987; Smith et al.,1995). In a PCR reaction, the primers 5′-TGCATGCATGGATCCATCGATTTTCGTTCG-3′ (SEQ ID NO: 2) and 5′-CGAGCTCGGTACCTGATTACCAATTAGAAT-3′(SEQ ID NO: 3), which contained NsiI and SacI restriction sites at theirrespective 5′-ends were used. The obtained PCR product was digested withNsiI and SacI and ligated into pGKV210 (Van der Vossen et al., 1987)that had been digested with SacI (partially) and NsiI. The resultingplasmid was designated pGKV210-spc. pE194 (Horinouchi and Weisblum,1982) was used as a template for the amplification of a promoterlesserythromycin-resistance gene. To do this, the primers5′-GGGTCGACCCTATAACCAAATTAAAGAGGG-3′ (SEQ ID NO: 4) and5′-CCCAAGCTTGGGCAGTTTATGCATCCCTTAAC-3′ (SEQ ID NO: 5) were used in a PCRreaction. These primers contained SalI and HindIII restriction sites attheir respective 5′-ends. The amplified fragment was digested with SalIand HindIII and the fragment was ligated into pGKV210-spc that had beendigested with SalI and HindIII. The resulting plasmid was designatedpIVS-E. To construct pIVS-PE, the promoter region of the mrp gene wasinserted into pIVS-E 5′ to the promoterless erythromycin-resistancegene. The promoter region of the mrp gene was amplified by PCR frompMRP11 (Smith et al., 1992) using the primers5′-CCCAAGCTTGGGAATTCATAATGTTTTTTTGAGG-3′ (SEQ ID NO: 6) and5′-GCGTCGACATCTACGCATAAAAAATCCCCC-3′ (SEQ ID NO: 7). These primerscontained EcoRI and SalI sites at their respective 5′-ends. AmplifiedDNA was digested with EcoRI and SalI and the resulting fragment wasligated into EcoRI and SalI-digested pIVS-E.

[0041] Construction of Genomic S. suis Libraries in pIVS-E.

[0042] Alu I partial digests of S. suis serotype 2 strain 10 DNA weresize fractionated (500-1000 bp) on a 0.8% (w/v) agarose gel. Thepurified fragments were ligated to SmaI and calf intestinal phosphatasedigested pIVS-E and the ligation mixtures were transformed to E. coliXL2-blue cells. Spectinomycin-resistant colonies were selected. Analysisof the transformants by PCR showed that more then 80% contained aninsert. From 15 pools of about 2000-3000 independent E. colitransformants, plasmid DNA was isolated. This plasmid DNA wassubsequently used for the electrotransformation of S. suis strain 10(Smith et al., 1995). This resulted in approximately 30,000 independentS. suis transformants. The transformants were pooled and stored at −80°C.

[0043] DNA Techniques.

[0044] Routine DNA manipulations and PCR reactions were performed asdescribed by Sambrook et al. (1989). DNA sequences were determined on a373A DNA Sequencing System (Applied Biosystems). Samples were preparedby using the ABI/PRISM dye terminator cycle sequencing ready reactionkit (Applied Biosystems). Custom-made sequencing primers were purchasedfrom Life Technologies. Sequencing data were assembled and analyzedusing the McMollyTetra software package. The BLAST program was used tosearch for protein sequences similar to the deduced amino acidsequences.

[0045] PCR reaction mixtures (50 μl) contained 10 mM Tris-HCl, pH 8.3,1.5 mM MgCl₂, 50 mM KCl, 0.2 mM of each of the four deoxynucleotidetriphosphates, 1 μM of each of the primers and 1 U of AmpliTaq Gold DNApolymerase (Perkin Elmer Applied Biosystems). DNA amplification wascarried out in a Perkin Elmer 9600 thermal cycler and the programincluded an incubation for ten minutes at 95° C. and 30 cycles of oneminute at 95° C., two minutes at 56° C. and two minutes at 72° C.

[0046] Assessment of Erythromycin Levels in Treated Piglets.

[0047] One-week-old specific pathogen-free (SPF) piglets were treatedorally with erythromycin stearate (Abbott, 20 or 40 mg body weight kg⁻¹)or intramuscularly with erythromycin (Erythrocin 200; Sanofi Sante, 20or 40 mg body weight kg⁻¹). Blood samples were collected 3 hours, 6hours or 24 hours after the administration of the antibiotics todetermine erythromycin levels.

[0048] Experimental Infections.

[0049] Gnotobiotic Great Yorkshire and Dutch Landrace crossed pigletswere obtained from sows by cesarian section. The surgery was performedin sterile flexible film isolators. The piglets were allotted to groups,each having 4 piglets, and were housed in sterile stainless steelincubators. Housing conditions and feeding regimens were as described(Vecht et al., 1989; Vecht et al., 1992). One-week-old piglets wereinoculated intravenously with S. suis strain 10 (pIVS-E), 10 (pIVS-PE)or 10 (pIVS-RE) as described (Vecht et al., 1989; Vecht et al., 1992,Table 3). Two hours after infection, the pigs were injectedintramuscularly with erythromycin for the first time and thereafterreceived erythromycin twice a day: once intramuscularly (Erythrocin, 40mg body weight kg⁻¹) and once orally (erythromycin stearate, 40 mg bodyweight kg⁻¹).

[0050] Piglets were monitored twice a day for clinical signs of disease,such as fever, nervous signs and lameness. Blood samples were collectedthree times a week from each pig. Leukocyte concentrations weredetermined using a conducting counter (Contraves A. G., Switzerland). Tomonitor infection with S. suis and to check for absence of contaminants,swabs of the nasopharynx and of feces were collected daily. The swabswere directly plated onto Columbia agar containing 6% (v/v) horse blood.After the piglets were killed, they were examined for gross pathologicalchanges. Tissue specimens were collected from the central nervoussystem, serosae, joints, lungs, heart and tonsils. The tissues werehomogenized in the presence of Todd-Hewitt medium using an Ultra-Turraxtissuemizer (Omni International) and frozen at −80° C. in the presenceof 15% (v/v) glycerol.

[0051] Results.

[0052] Promoter Selection System.

[0053] The plasmid pIVS-E was constructed to allow introduction of S.suis DNA fragments into a number of unique restriction sites in front ofa promoterless erythromycin-resistance resistance gene. The plasmidcarries the origin of replication of pWVO1, which functions in E. coliand in S. suis (Smith et al., 1995). S. suis strain 10 cells containingpIVS-E were sensitive to 1 μg erythromycin ml⁻¹ on agar plates. InpIVS-PE the promoter of the mrp gene of S. suis (Smith et al., 1992),which is highly expressed in vivo as well as in vitro, drives expressionof the erythromycin-resistance gene. S. suis strain 10 cells containingpIVS-PE were resistant to high concentrations of erythromycin (>256 μgerythromycin ml⁻¹) on agar plates. A S. suis DNA library in pIVS-E(pIVS-RE) was constructed and 30,000 individual S. suis clones ormutants were obtained. As determined by analysis of 24 randomly selectedtransformants, more than 80% of these clones or mutants contained aninsert (results not shown). Moreover, 2% of the clones were resistant to1 μg erythromycin ml⁻¹ on agar plates indicating the presence of somepromoter sequences that were functional in vitro.

[0054] Selection of Promoters Induced Under Iron-Restricted Conditions.

[0055] Gene sequences that were specifically induced on agar platesunder iron-restricted conditions were selected. For this purpose, about96,000 c.f.u. were plated under iron-limiting conditions on agar platescontaining deferoxamine mesylate and erythromycin. The 1500 coloniesthat grew on these plates were inoculated onto plates containingerythromycin, deferoxamine mesylate and FeSO₄. Twenty-four clones showedreduced growth in the presence of FeSO₄. The inserts of the 24 selectediri clones were amplified by PCR using primers complementary to the 5′ends of the erythromycin- and spectinomycin-resistance genes and thenucleotide sequences of these fragments were determined. The sequencedata showed that the 24 clones contained 18 unique sequences. The 18sequences were analyzed for similarity to known genes by comparison withthe sequences in the GenBank/EMBL and SWISSPROT databases. One sequence,iri3I, was identical to cps2A, a previously identified S. suis geneputatively involved in the regulation of capsule expression (Smith etal., 1999). Fourteen iri sequences were similar to sequences of known,non-S. suis, genes. Three of these sequences (iri2 (SEQ ID NO: 15),iri1, 6 and 22 (SEQ ID NO: 8), and iri34 (SEQ ID NO: 21) were similar tosequences of environmentally regulated genes previously selected byapplying the IVET to V. cholerae (Camilli and Mekalanos, 1995), S.aureus (Lowe et al., 1998) and P. aeruginosa (Wang et al., 1996),respectively. One, contained in iri1, 6, and 22 (SEQ ID NO: 8), wassimilar to the agrA gene of Staphylococcus aureus, a key locus involvedin the regulation of numerous virulence proteins. Three iri sequenceshad no significant similarity to any sequences in the databases (Table2).

[0056] Conditions for Selection of Promoter Sequences in Piglets.

[0057] To determine the antibiotic treatment regime required for asuccessful selection of in vivo-expressed promoter sequences, pigletswere treated with different concentrations of erythromycin once a day.The erythromycin was administered either orally or intramuscularly.Levels of erythromycin in sera were determined 3, 6 or 24 hours aftertreatment over one week. High erythromycin levels were detected threehours and six hours after both treatments (results not shown). However,24 hours after the treatments, the levels decreased dramatically. Basedon these data, we hypothesized that for efficient promoter selection, itwas necessary to treat the animals twice a day with erythromycin (40 mgkg⁻¹), once intramuscularly (at 9 a.m.) and once orally (at 4 p.m.).

[0058] To test this hypothesis, pigs were inoculated either with S. suisstrain 10 (pIVS-PE) or with strain 10 (pIVS-E). In pIVS-PE, the promoterof the mrp gene of S. suis (Smith et al., 1992), which is highlyexpressed in vivo as well as in vitro, drives expression of theerythromycin-resistance gene. The control plasmid, pIVS-E, does notcontain a promoter in front of the erythromycin-resistance gene. Thestrains were inoculated intravenously or intranasally. All pigs infectedwith strain 10 (pIVS-PE) showed specific S. suis symptoms (Table 3) and,except for one, all pigs died in the course of the experiment. Moreover,high numbers of bacteria were isolated from the central nervous system,the serosae and the joints. In contrast, none of the pigs inoculatedwith strain 10 (pIVS-E) showed specific clinical signs of disease andall survived the infection until the end of the experiment. Moreover,bacteria were not isolated from the central nervous system, the serosaeor the joints of these animals. These data demonstrated that invivo-expressed sequences could be selected from pigs using the appliedantibiotic treatment regimen.

[0059] Selection of Gene Sequences Expressed in Vivo in Piglets.

[0060] Piglets were inoculated intravenously with different doses (5×10⁵to 5×10⁸ c.f.u.) of the S. suis library (Table 3) and treated witherythromycin as described herein. Specific signs of disease developed inall animals three to eight days after infection (Table 3). High numbersof bacteria were recovered from tissues (central nervous system, joints,serosae, lung, liver, spleen, heart and kidney) of the individualpiglets. Analysis of the recovered bacteria showed that a limited numberof different clones were present in each of the bacterial samplesisolated from the diseased pigs. For example, 30 randomly selectedclones from the joints of one pig all possessed identical DNA inserts asassessed by PCR and DNA sequence analysis (results not shown). Inaddition, at 80% of the 62 sample sites analyzed, four randomly selectedclones were identical. However, from different tissues of a singleanimal, different clones or mutants could be isolated. On the otherhand, identical clones could be isolated from different, as well as fromcorresponding, tissues of different animals. These findings indicatedthat a limited number of clones had been selected in vivo and weregreatly enriched in the affected tissues. The observed selection was nottissue specific. Further, none of the selected clones failed to grow onagar plates that contained 1 μg erythromycin ml⁻¹.

[0061] Two-hundred forty-five clones were analyzed by PCR and partialsequence analysis. Among these, 22 unique ivs clones were found. The 22sequences were analyzed for similarity to sequences of known genes bycomparison with the GenBank/EMBL and SWISSPROT databases (Table 4). Thesequences of two genes showed similarity to genes encoding putativevirulence factors: ivs21, 26 and 30 which was identical to the epf gene,a previously identified S. suis gene, putatively involved in virulence(Smith et al., 1993; Smith et al., 1996); and ivs31 (SEQ ID NO: 37),which was similar to the fibronectin-binding protein of S. gordonii.Moreover, the sequences of two ivs genes (ivs25 (SEQ ID NO: 24) andivs6, 7, 13 and 14 (SEQ ID NO: 43)) were homologous to twoenvironmentally regulated ivi genes, previously identified using IVETselection in other bacterial species (Camilli and Mekalanos, 1995; Loweet al., 1998). Four ivs sequences (ivs25 (SEQ ID NO: 34); ivs23 and 24(SEQ ID NO: 33), ivs2, 4 and 28 (SEQ ID NO: 31); and ivs6, 7, 13 and 14(SEQ ID NO: 43)) were also found when the library was selected usingiron-restricted conditions. The remainder of the sequences showedsimilarity to sequences of known, non-S. suis genes, including two genesshowing similarity to mobile elements and five genes showing similarityto genes of unknown function.

[0062] The identification of environmentally regulated genes of S. suisserotype 2 by the use of iron-restricted conditions and by experimentalinfection of piglets is described. Eighteen unique iri genes and 22unique ivs genes were found. None of the ivs genes was exclusivelyexpressed in vivo. Four iri genes were identical to four clones selectedin vivo. The selected gene sequences encode for potential virulencefactors, expand our knowledge about the pathogenesis of S. suisinfections in pigs and are of value in control of the disease either bythe development of effective vaccines or by the development of newdiagnostic methods. A promoter trap was used to identify environmentallyregulated S. suis genes expressed under specific conditions, i.e.,during iron-restriction or during experimental infection. This systemdiffers from the antibiotic-based IVET system described for S.typhimurium (Mahan et al., 1995) in two ways. One is that the lacZreporter gene fusion is omitted in our vector constructions becauseinclusion of the lacZ gene resulted in structural instability of thevector. The other difference is that a plasmid system was used ratherthan a chromosomal integration system. A plasmid system was used becausethe low transformation efficiency of S. suis (Smith et al., 1995) mightprevent the generation of a complete gene library using a chromosomalintegration system.

[0063] From the data, it is evident that a number of inducible andenvironmentally regulated sequences were selected. Four iri genes wereidentical to four ivs genes. Because most bacteria require iron fortheir growth and because there is a limited amount of free ironavailable within the host (Payne, 1993), it might be expected that theexpression of some ivs genes is regulated by iron. With the in vivoselection system, tissue-specific colonization was not observed: clonesisolated from one piglet were also isolated from other piglets fromcorresponding as well as from different tissues. This might be due tothe mechanisms involved in the molecular pathogenesis of S. suisinfections in pigs. Furthermore, it was striking and different from theobservations made with IVET systems that only a limited number of clonescould be selected. In addition, we were not able to demonstrate that weselected for gene sequences that are exclusively expressed in vivo. Thiscould be explained either by the absence of promoter sequencesexclusively expressed in vivo among the 22 identified ivs genes, and/orby the inability of this plasmid-based system to identify such sequencesdue to gene dose effects.

[0064] A number of interesting genes were selected. Two ivs genes showedsimilarity to genes encoding putative virulence factors. Ivs21, 26 and30 were shown to be identical to the epf gene of S. suis (Smith et al.,1993), which is found in virulent strains of S. suis serotypes 1 and 2(Stockhofe-Zurwieden et al., 1996; Vecht et al., 1991; Vecht et al.,1992). Ivs31 (SEQ ID NO: 37) showed similarity to thefibronectin/fibrinogen-binding protein of S. gordonii (accession no.X65164) and group A Streptococci (Courtney et al., 1994). InStreptococci, fibronectin/fibrinogen-binding proteins play an importantrole in adhesion to host cells and are considered to be importantvirulence factors. The selection of these two ivs genes demonstrated theselectivity of the system and might be indicative for the relevance ofthe other ivs genes in the pathogenesis of S. suis infections in pigs.The performance of the system was further demonstrated by theobservation that two ivs genes, ivs25 (SEQ ID NO: 34) and ivs6, 7, 13and 14 (SEQ ID NO: 43) showed similarity to environmentally regulatedgenes previously identified using an IVET selection system in otherbacterial species.

[0065] Ivs25 (SEQ ID NO: 34) showed significant similarity to the sapRgene of S. mutans (accession no. P72485) and Lactobacillus sake Lb706(Axelsson and Holck, 1995) as well as to the agrA gene of S. aureus(Projan and Novick, 1997), both of which encode response regulatorproteins of bacterial two-component signal-transduction systems, thusmediating the response to an environmental signal (Projan and Novick,1997). Use of an IVET selection system for S. aureus in mice selectedthe region preceding the agrA gene, suggesting induction of agrAexpression under in vivo conditions (Lowe et al., 1998). Moreover, in S.aureus, the agr locus was shown to play an important role in alteringthe expression of a considerable number of virulence factors in responseto cell density (Projan and Novick, 1997).

[0066] Clones ivs6, 7, 13 and 14 (SEQ ID NO: 43) showed similarity to agene, iviVI, previously identified by IVET selection in V. cholerae(Camilli and Mekalanos, 1995). The function of iviVI is unknown.However, the genes showed similarity to members of the ATP-bindingcassette family of transporters. The sequenced portion of ivs6, 7, 13and 14 (SEQ ID NO: 43) included an N-terminal ATP-binding Walker A boxmotif, which is highly conserved in this transporter family.

[0067] Four ivs genes were identical to four iri genes. The first gene,ivs23 and 24 (SEQ ID NO: 33), which is identical to iri24 (SEQ ID NO:17), showed similarity to cpsY of S. agalactiae (Koskiniemi et al.,1998) and to oxyR of various organisms (Demple, 1999). CpsY of S.agalactiae is involved in the regulation of capsule expression andenvironmental induction of expression of the cpsY gene has beensuggested by Koskiniemi et al. (1998). In S. suis, ivs23 and 24 (SEQ IDNO: 33).and iri24 (SEQ ID NO: 17) are not linked to the capsular locus(Smith et al., 1999). The oxyR gene is the central regulator ofoxidative stress response in E. coli (Demple, 1999) and approximatelyten genes are under the control of the OxyR protein. The second gene,ivs2, 4 and 28 (SEQ ID NO: 31), which is identical to iri10 and 20 (SEQID NO: 9), showed similarity to the yoaE gene of E. coli (accession no.P76262), a putative ABC transporter protein. The third and the fourthgenes, ivs25 (SEQ ID NO: 34) and ivs6, 7, 13 and 14 (SEQ ID NO: 43) wereidentical to iri1, 6 and 22 (SEQ ID NO: 8) and iri2 (SEQ ID NO: 15),respectively. These genes also showed similarity to ivi genes selectedusing IVET in other bacterial species.

[0068] Based on data presented by Niven et al. (1999), selection of irigenes of S. suis is not expected. The authors described that S. suisdoes not require iron for growth. However, in their studies the authorsused media reduced from iron by using ethylenediaminedi-o-hydroxyphenylacetic acid (EDDA). Therefore, the differentconditions used in vitro may explain the different results obtained.

[0069] Two of the S. suis ivs genes, ivs1 (SEQ ID NO: 25) and ivs8 (SEQID NO: 44), showed similarity to transposon sequences. Moreover, one S.suis ivs gene, ivs2, 4 and 28 (SEQ ID NO: 31), had a GC% that wasconsiderably higher than the composition of the rest of the selectedgenes. It is striking that in S. typhimurium, several of the ivi clonesthat are required for full virulence have been found to be associatedwith mobile elements. Their atypical base composition and codon usagehas led to the suggestion that they have been acquired from otherbacterial species by horizontal transfer (Conner et al., 1998).

[0070] Our screen also identified five ivs genes that showed similarityto sequences encoding proteins of unknown function. These genes are notstandard housekeeping or metabolic genes.

[0071] Besides the four ivs/iri genes, a considerable number of otheriri genes have been selected in this study by plating the library underiron-restricted conditions. Interestingly, one of the selected irigenes, iri31, is identical to the cps2A gene of S. suis. This gene waspreviously isolated as a part of the capsular locus of S. suis serotype2 (Smith et al., 1999) and was implicated in the regulation of capsularpolysaccharide biosynthesis (Kolkman et al., 1997; Smith et al., 1999).Moreover, because the capsule of S. suis is expressed in larger sizeafter in vivo growth when compared to growth in vitro (Quessy et al.,1994), regulated expression of cps2A might be expected. Another irigene, iri7 (SEQ ID NO: 23), showed similarity to the rpgG gene of S.mutans. This gene was shown to be required for the biosynthesis ofrhamnose-glucose polysaccharide (Yamashita et al., 1999). Becauserhamnose is part of the polysaccharide capsule in S. suis serotype 2(Elliott and Tai, 1978), a role of the iri7 (SEQ ID NO: 23) gene incapsule biosynthesis can be proposed. Iri34 (SEQ ID NO: 21) showedsimilarity to the np 16 gene, previously identified using IVET selectionin P. aeruginosa and suspected to encode threonine dehydratase activity(Wang et al., 1996). Together with the observation that 4 iri genescould be selected by the in vivo approach, these data show that the irigenes encode important virulence factors for S. suis.

[0072] Contribution of Fibronectin-Binding Protein to Pathogenesis ofStreptococcus suis Serotype 2.

[0073]Streptococcus suis causes severe infections, such as meningitis,septicemia, and arthritis, in piglets. The animals often do not survivethe infection (6, 28). Occasionally, S. suis causes septicemia andmeningitis in humans (3). The pathogenesis of an S. suis infection israrely understood. Sows are symptomless carriers of S. suis on theirtonsils and pass the bacteria on to their piglets. The piglets cannotcope with the bacteria and subsequently develop the specific symptoms ofan S. suis infection. Until now, 35 capsular serotypes of S. suis havebeen described (26), but serotype 2 strains are most often isolated fromdiseased piglets. The capsule is an important virulence factor sincepiglets infected with an acapsular mutant of S. suis serotype 2 strainsdo not develop any clinical symptoms (22). Bacterial proteins have beensuggested to play a role in the pathogenesis as well (2, 26). Theexpression of murimidase-released protein (MRP), extracellular factor(EF) and suilysin was shown to be strongly associated with pathogenicstrains of S. suis serotype 2 (1, 29, 30). Since isogenic mutantslacking MRP and EF and isogenic mutants lacking suilysin were stillpathogenic in young piglets, these proteins are not absolutely requiredfor virulence (2, 23). Recently, a new virulence factor was identified(21) by using a complementation approach. The function of this virulencefactor in the pathogenesis has to be further investigated.

[0074] Many important virulence factors are environmentally regulatedand are induced at specific stages of the infection process (15). Toidentify these genes in S. suis, promoters and their downstreamsequences that are “on” during experimental S. suis infection of piglets(20) were cloned. Twenty-two in vivo selected (ivs) genes were found.Two of the ivs genes were directly linked to virulence since homologywas found to genes in the database that encode for known virulencefactors. One of these ivs genes (ivs-21) was identical to the epf geneof virulent S. suis serotype 2 strains (30). The other (ivs-31) (SEQ IDNO: 37) showed homology to genes encodingfibronectin-/fibrinogen-binding proteins of Streptococcus gordonii(GenBank accession no. X65164) and Streptococcus pyogenes FBP54 (8). Aconsiderable number of fibronectin-binding proteins of various bacterialspecies have been shown to be important virulence factors (12). In S.pyogenes, FBP54 was shown to be expressed in the human host and topreferentially mediate adherence to human buccal epithelial cells (7).It was shown that the FBP54 protein induces protective immunity againstS. pyogenes challenge in mice (13).

[0075] A fibronectin-/fibrinogen-binding protein of S. suis (FBPS) isdescribed herein and the sequence of fbps was determined. Bindingstudies showed that purified FBPS bound fibronectin and fibrinogen. Acontribution of FBPS to the pathogenesis of S. suis serotype 2 wasfound.

[0076] Materials and Methods.

[0077] Bacterial Strains and Growth Conditions.

[0078] The bacterial strains and plasmids used in this study are listedin Table 5. S. suis strains were grown in Todd-Hewitt broth (code CM189; Oxoid, Ltd.) and plated on Columbia blood base agar plates (codeCM331; Oxoid, Ltd., London, United Kingdom), containing 6% (vol/vol)horse blood. E. coli strains were grown in Luria Broth (17) and platedon Luria Broth containing 1.5% (wt/vol) agar. If required, antibioticswere added at the following concentrations: 50 μg/ml of spectinomycin(Sigma, St. Louis, Mo.) for E. coli and 100 μg/ml for S. suis, 100 μg/mlof ampicillin (Boehringer, Mannheim, Germany) for E. coli and 25 μg/mlof kanamycin (Boehringer) for E. coli.

[0079] DNA Techniques and Sequence Analysis.

[0080] Routine DNA manipulations were performed as described by Sambrooket al. (19). DNA sequences were determined on a 373A DNA SequencingSystem (Applied Biosystems, Warrington, Great Britain). Samples wereprepared by use of an ABI Prism dye terminator cycle sequencing readyreaction kit (Applied Biosystems). Sequencing data were assembled andanalyzed using the Lasergene program (DNASTAR). The BLAST softwarepackage was used to search for protein sequences homologous to thededuced amino acid sequences in the GenBank/EMBL databases.

[0081] Southern Blotting and Hybridization.

[0082] Chromosomal DNA was isolated as described by Sambrook et al.(19). DNA fragments were separated on 0.8% agarose gels and transferredto GeneScreen Plus hybridization transfer membrane (NEN™ Life ScienceProducts, Boston, USA) as described by Sambrook et al. (19). DNA probesof the fbps and spc genes were labeled with (α-³²P)dCTP (3,000 Ci/mmol;Amersham Life Science, Buckinghamshire, Great Britain) by use of arandom primed DNA labeling kit (Boehringer). The DNA on the blots waspre-hybridized for at least 30 minutes at 65° C. and subsequentlyhybridized for 16 hours at 65° C. with the appropriate DNA probes in abuffer containing 0.5 M sodium phosphate (pH 7.2), 1 mM EDTA and 7%sodium dodecyl sulphate. After hybridization, the membranes were washedtwice with a buffer containing 40 mM sodium phosphate (pH 7.2), 1 mMEDTA and 5% sodium dodecyl sulphate for 30 minutes at 65° C. and twicewith a buffer containing 40 mM sodium phosphate (pH 7.2), 1 mM EDTA and1% sodium dodecyl sulphate for 30 minutes at 65° C. The signal wasdetected on a phosphor-imager (Storm; Molecular Dynamics, Sunnyvale,Calif.).

[0083] Construction of a fbps Knock-Out Mutant.

[0084] To construct the mutant strain 10ΔFBPS, the pathogenic strain 10(27, 29) of S. suis serotype 2 was electrotransformed (24) with theplasmid pFBPS7-47. In this plasmid, the fbps gene was inactivated by theinsertion of a spectinomycin-resistance gene. To create pFBPS7-47 (FIG.1), the 382 bp SalI-SalI fragment of pFBPS7-46 was replaced by the 1.2kb EcoRV-SmaI fragment of pIC-Spc, containing the spectinomycinresistance gene, after the SalI sites of the vector were made blunt(FIG. 1). After electrotransformation of strain 10 with pFBPS7-47,spectinomycin-resistant colonies were selected on Columbia agar platescontaining 100 μg/ml of spectinomycin. Southern blotting andhybridization experiments were used to select for double cross-overintegration events (data not shown).

[0085] FBPS Expression Construct.

[0086] To construct an FBPS expression plasmid, the QIAexpress Kit(Qiagen GmbH, Hilden, Germany) was used. The primers corresponded topositions 250 to 272 and from 1911 to 1892 of the fbps gene. Thesequences of these primers were5′(GCGGATCCGATGACGATGACAAATCTTTTGACGGATTTTTTTTAC)3′ (SEQ ID NO: 46) and5′(CCCAAGCTTGGGCATGAACTAGATTTTCATGG)3′ (SEQ ID NO: 47). The primerscontained restriction sites for BamHI and HindIII, respectively, toamplify the fbps gene from pFBPS7-47. The amplified PCR product wasdigested with BamHI and HindIII and the 1.8 kb fbps gene was cloned intopQE-30 digested with BamHI and HindIII, yielding pQE-30-FBPS.pQE-30-FBPS was transformed to M15 (pREP4).

[0087] Purification of FBPS.

[0088] M15 (pREP4) (pQE-30-FBPS) was used to express and purify the FBPSusing the QIAexpressionist™ (Qiagen). In short, M15 (pREP4) (pQE-30FBPS)cells were grown exponentially; 1 mM IPTG was added and the cells wereallowed to grow another four hours at 37° C. Subsequently, cells wereharvested and lysed. The cleared supernatants were loaded onto Ni²⁺-NTAagarose columns. FBPS containing a 6×HIS tag was bound to theNi²⁺⁺-column. The columns were washed and the protein was eluted.Different buffers were used for native and for denaturing purification.FBPS purified under denaturing conditions was renaturated on a Ni²⁺-NTAcolumn by using a linear 6 M-1 M urea gradient in 500 mM NaCl, 20%glycerol and 20 mM Tris-HCl (pH7.4), containing protease inhibitors (25μg/ml of pefabloc, 0.7 μg/ml of pepstatin, 1 μg/ml of aprotinin, 0.5μ/ml of leupeptin). All procedures were performed according to themanufacturer's recommendations. The 6×HIS tag was removed from theprotein by incubating purified FBPS in 20 mM Tris-HCl (pH 7.4), 50 mMNaCl, 2 mM CaCl₂ and 0.5 U of light chain enterokinase (New EnglandBiolabs, Beverly, Mass.) for 16 hours at RT.

[0089] Immunization of Rabbits with FBPS.

[0090] Purified and renaturated FBPS was used to immunize two rabbits.To remove urea, the protein was dialyzed against phosphate bufferedsaline (136 mM NaCl; 2.68 mM KCl; 8.1 mM Na₂HPO₄; 2.79 mM KH₂PO₄ (pH7.2)) over night at 4° C. Seven days before immunization, blood wascollected from the rabbits to determine the natural titers against FBPS.At day one, those rabbits with negative anti-FBPS titers were immunizedintramuscularly with two times 0.5 ml of 100 μg/ml of FBPS in awater-in-oil emulsion (Specol; ID-Lelystad). At day 28, rabbits wereimmunized for the second time using the same amount of protein and thesame route of immunization. Three weeks after the second immunization,the rabbits were sacrificed and blood was collected. The blood wascoagulated and serum was collected and used for immunodetection of FBPS.

[0091] Immunodetection of FBPS.

[0092] Proteins were separated by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) by standard procedures(19). Proteins in the gel were visualized using SYPRO-orange (MolecularProbes, Sunnyvale, Calif.) staining according to the manufacturer'srecommendations. Signals were detected on a phosphor imager (Storm;Molecular Dynamics). A known bovine serum albumin concentration rangewas used as a standard to calculate the amounts of protein present inthe gel. The Molecular Dynamics program was used for the calculations.

[0093] Proteins were transferred to a nitrocellulose membrane bystandard procedures (19). The membranes were blocked in Blotto:Tris-buffered saline (TBS) (50 mM Tris-HCl (pH 7.5), 150 mM NaCl)containing 4% skimmed milk and 0.05% Tween 20 at room temperature (RT)for one hour. To detect recombinant purified FBPS, membranes wereincubated with a monoclonal antibody against the 6×HIS tag (Clontech,Palo Alto, Calif.) in a 1:10,000 dilution in Blotto-TBS (1:1) at RT forone hour, followed by an incubation with alkaline phosphatase-conjugatedanti-mouse antibody in a 1:1,000 dilution in Blotto-TBS (1:1) at RT forone hour. Reactivity of purified FBPS was tested by using a convalescentserum of a pig that had survived an S. suis infection. Nitrocellulosemembranes were incubated with the polyclonal pig serum in a 1:200dilution in Blotto-TBS (1:1) at RT for one hour, followed by incubationat RT for one hour with alkaline phosphatase-conjugated anti-swineantibody in a 1:2,000 dilution in Blotto-TBS (1:1). As a substrate,Nitro Blue Tetrazolium (Merck, Darmstadt, Germany) bromochloroindolylphosphate (Sigma) was used. All washing steps were performed inBlotto-TBS (1:1).

[0094] Fibronectin and Fibrinogen Binding.

[0095] Binding studies were performed by indirect Western blotting.Proteins were separated by SDS-PAGE and transferred to a nitrocellulosemembrane as described herein. The membranes were blocked in MPBS: PBScontaining 4% skimmed milk and 0.05% Tween 20. Subsequently, themembrane was incubated with 5 μg/ml of human fibronectin (fn) (Sigma) or5 μg/ml of human fibrinogen (fgn) (Sigma) in PBS containing 5% fetalcalf serum, 2% NaCl, and 0.05% Tween 80 at RT for one hour. To detectbound fibronectin and fibrinogen, the membranes were incubated withhorse-radish peroxidase-conjugated anti-fibronectin (DAKO) oranti-fibrinogen (DAKO) antibodies in a 1:1,000 dilution in PBScontaining 5% fetal calf serum, 2% NaCl, and 0.05% Tween 80 at RT forone hour. The signal was visualized by using ECL⁺ (Amersham PharmaciaBiotech, N.J.) according to the manufacturer's recommendations. Signalswere detected on a phosphor imager (Storm; Molecular Dynamics). Allwashing steps were performed in MPBS-PBS (1:1).

[0096] Experimental Infections.

[0097] Germ-free piglets, cross-breeds of Great Yorkshire and DutchLandrace, were obtained from sows by cesarean section. The surgery wasperformed in sterile flexible film isolators. Piglets were allotted togroups of four and were housed in sterile stainless steel incubators.Housing conditions and feeding regimens were as described (27, 29).Six-day-old piglets were inoculated intranasally with about 10⁷ cfu ofBordetella bronchiseptica 92932 to predispose the piglets to infectionwith S. suis. Two days later, the piglets were inoculated intranasallywith 10⁶ cfu of S. suis strain 10 plus 10⁶ cfu of S. suis strain10ΔFBPS. To determine differences in virulence between wild-type andmutant strains, LD₅₀ values should be determined. To do this, largenumbers of piglets are required. For ethical reasons, this is notacceptable. To circumvent this problem, co-colonization studies wereperformed.

[0098] To monitor for the presence of S. suis and B. bronchiseptica andto check for absence of contaminants, swabs taken from the nasopharynxand the feces were cultured three times a week. The swabs were plateddirectly onto Columbia agar containing 6% horse blood or grown for 48hours in Todd-Hewitt broth and subsequently plated onto Columbia agarcontaining 6% horse blood. Pigs were monitored twice a day for clinicalsigns and symptoms, such as fever, nervous signs, and lameness. Bloodsamples from each pig were collected three times a week. Leukocytes werecounted with a cell counter. The piglets were killed when specific signsof an S. suis infection were observed, such as arthritis or meningitis,or when the pigs became mortally ill. The other piglets were killed twoweeks after inoculation with S. suis and examined the same way as thepiglets that were killed based on their clinical symptoms. All pigletswere examined for pathological changes.

[0099] Tissue specimens from heart, lung, liver, kidney, spleen, andtonsil, and from the organs specifically involved in an S. suisinfection (central nervous system (CNS), serosae, and joints) weresliced with a scalpel or a tissuenizer. Tissue slices from each organ orsite were resuspended in 2-25 ml of Todd-Hewitt containing 15% glyceroldepending on the size of the tissue slice. The suspension wascentrifuged at 3,000 rpm for five minutes. The supernatant was collectedand serial dilutions were plated on Columbia agar containing 6% horseblood, as well as on Columbia agar plates containing 6% horse blood and100 μg/ml of spectinomycin to quantitate the number of wild-type andmutant bacteria present. The number of mutant strain 10ΔFBPS cells wasdetermined by counting the number of CFU on the appropriate serialdilution on the selective plates; the number of wild-type strain 10cells was determined. by counting the number of CFU on the appropriateserial dilution on the Columbia Agar blood plates of which the number ofCFU counted on the selective plates was subtracted. When wild-type andmutant bacteria were found in tissues, the ratio of wild-type and mutantstrain was determined again by toothpicking about 100 individualcolonies onto both Columbia Agar plates and onto Columbia Agar platescontaining 100 μg/ml spectinomycin.

[0100] All animal experiments were approved by the ethical committee ofthe Institute for Animal Science and Health in accordance with the Dutchlaw on animal experiments.

[0101] Nucleotide Sequence Accession Number.

[0102] The nucleotide sequence data fbps have been submitted to GenBank,in which the sequence is listed under accession no. AF438158.

[0103] Results.

[0104] Cloning of the S. suis fbps Gene.

[0105] One of the in vivo selected genes (ivs-31) (SEQ ID NO: 37) (20)showed homology to the 5′ part of genes encoding for FlpA and FBP54,fibronectin-binding proteins (FnBP) of Streptococcus gordonii (GenBankaccession no. X65164) and Streptococcus pyogenes (8), respectively. Toclone the entire fbps gene of S. suis, ivs31 (SEQ ID NO: 37) was used asa probe to identify a chromosomal DNA fragment of S. suis serotype 2containing flanking fbps sequences. A 5 kb EcoRI fragment was identifiedand cloned in pGEM7Zf(+) yielding pFBPS7-46 (FIG. 1). Sequence analysisrevealed that this fragment contained the entire fbps gene of S. suisserotype 2.

[0106] An open reading frame of 1659 bp coding for a polypeptide of 553amino acids was found. The putative ATG start codon is preceded by asequence similar to ribosome binding sites of gram-positive bacteria.Further upstream, two putative promoter sequences could be identified.Upstream of these promoter sequences of fbps, a direct repeat was foundthat could serve as a transcription terminator of the gene located 5′ offbps. Downstream of fbps, a gene that showed homology to analpha-acetolactate decarboxylase was found. This gene is transcribed inthe opposite direction of fbps. The deduced amino acid sequence wasaligned with that of several previously identified FnBPs from otherbacteria. As expected, FBPS was substantially homologous to FlpA of S.gordonii (76%) and also showed homology to FnBP's of other organisms,like Streptococcus pneumoniae (73%), S. pyogenes (69%), Lactococcuslactis (59%), and Bacillus subtilis (41%). Compared to the sequence ofFBP54, FBPS has a longer N-terminus with 76 additional amino acids. Thislonger N-terminus was also seen in other organisms like S. gordonii, S.pneumoniae and B. subtilis. In FBP54, the primaryfibronectin-/fibrinogen-binding domain was localized to its N-terminalpart, to the first 89 amino acids (8). Over this region, the homology ofFBPS to FBP54 is very high (80%), suggesting that FBPS can bind bothfibronectin and fibrinogen.

[0107] Binding of FBPS to Fibronectin and Fibrinogen.

[0108] To confirm the binding of FBPS of S. suis to fibronectin (fn) andfibrinogen (fgn), FBPS was purified under native conditions. A proteinexpression construct, which expresses FBPS with a 6×HIS tag fused to theN-terminus, was used for purification. Four hundred μg of FBPS waspurified from 50 ml of exponential-phase E. coli cells after inductionwith IPTG. The purity of this FPBS was determined with SDS-PAGE andWestern blotting (FIG. 2). The induced E. coli lysate contained a broadrange of proteins, among which the 64 kDa protein FBPS was present(panel A, lane 1). After purification, highly purified FBPS with 6×HIStag was obtained (panel A, lane 2). When both samples were incubatedwith a monoclonal antibody against the 6×HIS tag, FBPS was the onlyprotein that was detected (panel B).

[0109] To determine whether FBPS binds fn and fgn, a Western blotcontaining purified FBPS was incubated with soluble human fn and humanfgn (FIG. 3, panels A and B). Specific binding of fn and fgn to FBPS wasdetected. No binding of fn and fgn to BSA, a negative control protein,was observed. To exclude possible background signals due toimmunoglobulin-binding of FBPS, the same experiment was performedwithout addition of fibronectin or fibrinogen. No binding was found(FIG. 3, panels C and D) indicating that the binding was specific forfibronectin and fibrinogen. To control whether the binding of fn and fgnto FBPS was not mediated by the 6×HIS tag, the tag was removed by anenterokinase treatment. FIG. 3, panels E and F, show that FBPS withoutthe 6×HIS tag still efficiently bound to fn and fgn. Therefore, itappears that FBPS can specifically bind to fn and fgn.

[0110] Immunogenicity of FBPS.

[0111] Since it was shown that FBP54 induced a protective immuneresponse in mice against a lethal dose of S. pyogenes (13), it wasdetermined whether purified FBPS was recognized by convalescent serum ofa pig that survived an S. suis infection. As shown in FIG. 2 panel C,the FBPS reacted with this anti-serum. When the same experiment wasperformed with non-immune serum of an SPF piglet, no band of the size ofFBPS was detected (data not shown). These findings indicate that FBPS isexpressed in vivo and that the protein is indeed immunogenic in youngpigs.

[0112] Distribution of the fbps Gene Among the 35 S. suis Serotypes.

[0113] Since we were interested in a cross-protective vaccine candidate,the presence of the fbps gene among the various S. suis serotypes wasanalyzed. Ivs-31 (SEQ ID NO: 37), the clone containing the promoter andthe 5′-part of the fbps gene, was radiolabeled and chromosomal DNA ofthe reference strains of the 35 different S. suis serotypes washybridized with this probe. The three different phenotypes of S. suisserotype 2, a pathogenic, a non-pathogenic and a weak pathogenic strain,were included in this study. The fbps gene was present in all S. suisserotypes and phenotypes except for serotypes 32 and 34 (FIG. 4).

[0114] Role of FBPS in Pathogenesis.

[0115] To test the role of FBPS in the pathogenesis of S. suis, anisogenic knock-out mutant of FBPS was constructed in strain 10, strain10ΔFBPS. Since upstream of fbps a direct repeat was found that couldserve as a transcription terminator and downstream of fbps a geneshowing homology to an alpha-acetolactate decarboxylase was found thatis transcribed in the opposite direction, polar effects to genesupstream or downstream of fbps are not expected. To verify that themutant strain 10ΔFBPS did not produce FBPS, protoplasts of strain 10 andstrain 10ΔFBPS were subjected to SDS-PAGE and Western blotting. FBPS wasdetected using a polyclonal antiserum raised against purified FBPS. Itwas shown that strain 10ΔFBPS expressed no FBPS, while strain 10 didexpress FBPS (data not shown). Subsequently, the virulence of thismutant strain was tested in an experimental infection in piglets.

[0116] The mutant strain 10ΔFBPS was used in a competition challengeexperiment with the wild-type strain to determine the relativeattenuation of the mutant strain. Using in vitro conditions, the growthrates of the wild-type and mutant strain in Todd-Hewitt medium werefound to be essentially identical (data not shown). Wild-type and mutantstrain were inoculated at an actual ratio of 0.65 (1.63×10⁶ cfu ofwild-type bacteria ml⁻¹ and 3.09×10⁶ CfU of mutant bacteria ml⁻¹).During the experiment, piglets that developed specific S. suis symptoms(meningitis, arthritis, or mortal illness) were killed. Piglets that didnot develop these symptoms were killed at the end of the experiment.From all piglets, the ratio of wild-type and mutant strain in variousorgans was determined.

[0117] As shown in FIG. 5, panel A, similar numbers of wild-type andmutant bacteria were re-isolated from tonsils. The ratio was similar tothe input ratio (ratio varied from 0.33-0.85, average 0.61). Thisindicates that the efficiency of colonization of wild-type and mutantstrain on tonsils was essentially identical. Apparently, FBPS is notstrictly required for colonization of the tonsils of the piglets. Threeout of four piglets developed clinical signs specific for an S. suisinfection. Two piglets (4664 and 4666) showed clinical signs ofarthritis and one piglet (4668) showed clear central nervous signs. Thefourth piglet did not develop any clinical signs. These observationscoincided with pathomorphological abnormalities of the specific organsof an S. suis infection in post-mortem sections.

[0118] As shown in FIG. 5, panel B, exclusively wild-type bacteria werere-isolated from the joints of piglet 4664 and from the CNS of piglet4668. The numbers of CFU of wild-type bacteria that were re-isolatedfrom these specific organs were very high, while no mutant bacteria werefound. From the joints of pig 4666, low numbers of both wild-type andmutant bacteria were re-isolated in a ratio of 0.84 (1.0×10² CFU ofwild-type bacteria and 5.2×10² CFU of mutant bacteria), a ratioessentially identical to the input ratio (FIG. 5, panel B). Southernblot experiments using the fbps and the spc genes as probes, confirmedthat the mutant bacteria isolated from the joint of pig 4666 were indeedidentical to the input mutant bacteria. Taken together, these dataindicate, that the FBPS mutant is capable of reaching and colonizing thespecific S. suis organs (at least the joints), but that the mutant isfar less efficiently recovered from organs than the wild type. TABLE 1Bacterial strains and plasmids. Strain/plasmid relevant characteristics*source/reference Strain E. coli XL2 blue Stratagene S. suis 10 virulentserotype 2 strain (Vecht et al., 1992) Plasmid pKUN19-spc replicationfunctions pUC, Amp^(R), (Konings et al., Spc^(R) 1987, Smith et al.,1995) pGKV210 replication functions pWVO1, Cm^(R), (Van der VossenEm^(R) et al., 1987) pE194 Em^(R) (Horinouchi & Weisblum, 1987) pMR11pKUN19 containing S. suis mrp gene (Smith et al., 1992) pIVS-Ereplication functions pWVO1, Spc^(R), this work promoterless emR gene ofpE194 pIVS-PE pIVS-E containing promoter of mrp this work preceding thepromoterless emR gene pIVS-RE pIVS-E containing random S. suis this worksequences preceding the promoterless emR gene

[0119] TABLE 2 Iron-restriction induced S. suis genes. Insert Data basehomology % Clone (bp) GC % (accession no.) Function of homolog IdentityRegulatory functions iri 1, 6, 22 800 34 S. mutans SapR (U75483)response regulator protein 44 (SEQ ID NO: 8) S. aureus AgrA (X52543)response regulator protein 51 S. aureus Ivi2 iri 24 850 38 S. agalactiaeCpsY (CAB36982) regulation capsule expression 46 (SEQ ID NO: 17) E. coliOxyR (P11721) oxidative stress regulator 51 iri 23 1000 38 B. subtilisYvyD (P28368) sigma-54 modulator homologue 44 (SEQ ID NO: 16) Metabolicfunctions iri 7 800 39 S. mutans RgpG (Q9XDW8) rhamnose-glucosebiosynthesis 76 (SEQ ID NO: 23) iri 11 700 34 L. lactis NrdD (Q9ZAX6)anaerobic ribonucleotide reductase 51 (SEQ ID NO: 10) iri 14 500 38 S.pneumoniae SulB (Q54614) dihydrofolate synthetase 41 (SEQ ID NO: 12) iri16 850 48 B. subtilis TrmU (O35020) RNA methyltransferase 62 (SEQ ID NO:13) iri 32 300 41 C. histolyticum RuvB (O9ZNJ5) hypoxanthine-guanine 55(SEQ ID NO: 20) phosphoribosyl transferase iri 34 1000 44 L. lactis IlvA(U92974) probable threonine dehydratase 56 (SEQ ID NO: 21) P. aeruginosaPn16 Transporter functions iri 2 750 36 B. subtilis YloD (O34328)putative guanylate kinase 50 (SEQ ID NO: 15) S. gordonii ComYA (U81957)putative ABC transporter 37 Vibrio cholerae IviVI (Q56605) putative ABCtransporter 47 iri 10, 20 1350 51 E. coli YoaE (P76262) putativetransport protein 94 (SEQ ID NO: 9) Unknown iri 13, 15, 27 800 34 M.tuberculosis MTCY336_33 unknown 38 (SEQ ID NO: 11) hypothetical protein(O06593) iri 29 850 36 S. aureus Yp15 (P13977) unknown 39 (SEQ ID NO:18) hypothetical protein iri 18 800 39 S. crista hypothetical protein(AAF61316) unknown 82 (SEQ ID NO: 14) iri 3 700 36 no homology found(SEQ ID NO: 19) iri 4 700 36 no homology found (SEQ ID NO: 22) iri 8, 26900 35 no homology found (SEQ ID NO: 24)

[0120] TABLE 3 Virulence of S. suis 10 (pIVET-E), 10 (pIVET-PE) and 10(pIVET-RE) in gnotobiotic piglets. Clinical index of the group ¦ Non-Fever No. of pigs from which Strains/ No. of Dose (route MortalityMorbidity Specific specific index Leukocyte S. suis was isolated librarypiglets of infection) * (%) ¥ (%) ∥

à index £ CNS Serosae Joints 10 (pIVS-E) 4 10⁶ (i.n.) 0 0 0 6 9 75 0 0 010 (pIVS-E) 4 10⁶ (i.v.) 0 0 6 12 31 0 0 0 0 10 (pIVS-PE) 4 10⁶ (i.n.)100 100 30 40 35 100 3 0 2 10 (pIVS-PE) 4 10⁶ (i.v.) 75 100 50 42 43 503 3 4 10 (pIVS-RE) 4 5 × 10⁵ (i.v.) 100 100 56 75 44 83 2 2 4 10(pIVS-RE) 4 5 × 10⁶ (i.v.) 100 100 43 73 43 60 3 0 4 10 (pIVS-RE) 4 5 ×10⁷ (i.v.) 100 100 60 74 48 75 4 1 4 10 (pIVS-RE) 4 5 × 10⁸ (i.v.) 100100 49 70 37 50 3 3 4

[0121] TABLE 4 S. suis genes selected in pigs. Sites of Insert Data basehomology Function % Clone isolation (bp) GC % (accession no.) of homologIdentity Putative virulence factors ivs 31 CNS 200 47 S. gordonii FlpA(X65164) fibronectin/fibrinogen binding 70 (SEQ ID NO: 37) Regulatoryfunctions ivs 25 joint 800 34 S. mutans SapR (P72485) response regulatorprotein 49 (SEQ ID NO: 34) S. aureus AgrA (X52543) response regulatorprotein 51 S. suis Iri 1, 6, 22 100 ivs 23, 24 other 850 38 S.agalactiae CpsY (CAB36982) regulation capsule expression 46 (SEQ ID NO:33) E. coli OxyR (P11721) oxidative stress regulator 51 S. suis Iri 24100 ivs 16 CNS 800 43 S. epidermidis AltR (U71377) putativetranscriptional regulator 26 (SEQ ID NO: 28) ivs 20 lung 800 41 L.lactis AldR (O34133) putative regulator AldR 64 (SEQ ID NO: 32)Metabolic functions ivs 33 CNS 570 36 E. coli ThrC (P00934) threoninesynthase 41 (SEQ ID NO: 39) ivs 5, 10, CNS, joint 900 36 S. gordonii Tdk(P47848) thymidine kinase 87 12, 22 (SEQ ID NO: 42) ivs 18 lung 730 32S. mutans NADH oxidase (JC4541) NADH oxidase 80 (SEQ ID NO: 29)Transporter functions ivs 2, 4, 28 CNS, joint 1350 51 E. coli YoaE(P76262) putative transport protein 94 (SEQ ID NO: 31) S. suis iri 10,20 100 ivs 3 joint 1000 42 S. mutans OrfU (AF267498) putative ABCtransporter (permease) 33 (SEQ ID NO: 36) ivs 6, 7, CNS, joint 1350 36B. subtilis Ylo D (O34328) putative guanylate kinase 50 13, 14 (SEQ IDNO: 43) S. gordonii ComYa (U81957) putative ABC transporter 37 V.cholera IviVI (Q56605) putative ABC transporter 47 S. suis Iri 2 100Transposases ivs 8 CNS 600 41 S. pneumoniae transposase transposase 70(SEQ ID NO: 44) (Z86112) ivs 1 joint 1600 39 C. perfringens (X71844)putative transposase 56 (SEQ ID NO: 25) Miscellaneous ivs 32, 35 CNS 50038 S. typhimurium FliF (P15928) flagellar M-protein precursor 36 (SEQ IDNO: 38) ivs 9, 17 joint, CNS 800 36 B. subtilis ComE ORF2 (P32393)competence development 37 (SEQ ID NO: 45) ivs 11 serosea 800 44 P.syringae TabA (P31851) diaminopimelate decarboxylase/ 53 (SEQ ID NO: 26)tabtoxin Unknown ivs 15 CNS 750 42 B. subtilis conserved hypotheticalunknown 43 (SEQ ID NO: 27) protein YdiB (D88802) ivs 29 joint 800 38 S.salivarius hypothetical protein unknown 79 (SEQ ID NO: 35) (AF130465)ivs 34 CNS 600 43 B. subtilis conserved hypothetical unknown 61 (SEQ IDNO: 40) protein YRRK (O34634) ivs 36 joint 830 42 B. subtilishypothetical protein unknown 35 (SEQ ID NO: 41) YqeG (P54452) ivs 19lung 950 34 S. cristatus hypothetical protein unknown 86 (SEQ ID NO: 30)(U96166)

[0122] TABLE 5 Bacterial strains and plasmids. Strain/plasmid RelevantCharacteristics^(a) Source/reference Strains E. coli XL2-Blue recA1endA1 gyrA96 thi-1 hsdR17 supE44 relA1 Stratagene lac (F^(l) proABlacI^(q) Z)M15 TN10 (Tet^(R)) amy Cm^(R)) M15 Nal^(S) Str^(S) Rif^(S)Thi⁻ Lac⁻ Ara⁺ Gal⁺ Mtl⁻ F⁻ RecA⁺ Uvr⁺ Lon⁺ Qiagen S. suis 10 Virulentserotype 2 strain Vecht et al. (29) 10ΔFBPS Isogenic fbps mutant ofstrain 10 This work Plasmids pGEM7Zf(+) Replication functions pUC,Amp^(R) Promega Corp. pKUN19 Replication functions pUC, Amp^(R) Koningset al. (14) pIC19R Replication functions pUC, Amp^(R) Marsh et al. (16)pDL282 Replication functions of pBR322 and pVT736-1, Amp^(R), Spc^(R)Sreenivasan et al. (25) pIC-spc pIC19R containing Spc^(R) gene of pDL282Lab collection pQE-30 Replication functions pBR322, Amp^(R), expressionvector, 6x HIS tag Qiagen pQE-30-FBPS pQE-30 containing the 1.8 kb fbpsgene This work pREP4 Replication functions pACYC, Kan^(R), lacI geneQiagen pE194 Em^(R) Horinouchi and Weisblum (11) pIVS-E Replicationfunctions of pWVO1, Spc^(R), promoterless erm gene of pE194 Smith et al.(20) pIVS-31 pIVS-E containing 200 bp showing homology to Streptococcusgordonii Smith et al. (20) flpa pFBPS7-46 pGEM7Zf(+) containingEcoRI-EcoRI fragment of fbps This work pFBPS7-47 pFBPS7-46 in which 382bp SalI-SalI fragment is replaced by 1.2 kb Spc^(R) This work frompIC-spc

[0123] TABLE 6 Numbers of re-isolated wild-type (strain 10) and mutant(strain 10ΔFBPS) bacteria from organs of infected piglets (mean actualinoculation ratio 65% mutant strain). Pig number 4664 4665 4666 4667perc.^(c) perc.^(c) perc.^(c) perc.^(c) Organ w.t.^(a) mut.^(b) mut.w.t.^(a) mut.^(b) mut. w.t.^(a) mut.^(b) mut. w.t.^(a) mut.^(b) mut.Tonsil 1.77^(e)5 3.29^(e)5 65 4.35^(e)5 2.42^(e)6 85 5.34^(e)4 8.73^(e)461 7.94^(e)5 3.96^(e)5 33 pus joint 1 6.75^(e)4 <10 0 1.02^(e)2 5.2^(e)284 pus joint 2 5.15^(e)4 <10 0 CNS 1.88^(e)5 <10 0

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1 47 1 6 PRT Artificial Sequence Cell wall signalling sequence 1 Leu ProXaa Thr Gly Glu 1 5 2 30 DNA artificial sequence PCR primer containingNsiI and SacI restriction sites at the 5′ end 2 tgcatgcatg gatccatcgattttcgttcg 30 3 30 DNA artificial sequence PCR primer containing NsiIand SacI restriction sites at the 5′-end 3 cgagctcggt acctgattaccaattagaat 30 4 30 DNA artificial sequence PCR primer containing SalIand HindIII restriction sites at the 5′-end 4 gggtcgaccc tataaccaaattaaagaggg 30 5 32 DNA artificial sequence PCR primer containing SalIand HindIII restriction sites at the 5′-end 5 cccaagcttg ggcagtttatgcatccctta ac 32 6 34 DNA artificial sequence PCR primer containingEcoRI and SalI restriction sites at the 5′-end 6 cccaagcttg ggaattcataatgttttttt gagg 34 7 30 DNA artificial sequence PCR primer containingEcoRI and SalI restriction sites at the 5′-end 7 gcgtcgacat ctacgcataaaaaatccccc 30 8 335 DNA Streptococcus suis iri 1, 6, 22 8 gaaacgtcagtaaggtataa attcctagaa gttnttggta aaccaaatca atnattggaa 60 tcaattggggaagcagggaa tcatcaattt ttctttttag atattgaaat aaaaggagaa 120 gaaaagaaaggaatggaaat cgctaaagaa atccgggctc gagatcctta tgctgctatt 180 gtctttgtaacaactcactc agaattnatg ccagtaacat atcgttatca ggtttctgct 240 ttagattttatagataaagg cctggaggat cgtgactttc aaaaggcagt atcagangtc 300 ttagtgcatgcttttgaaaa tatcgatcat actnt 335 9 347 DNA Streptococcus suis iri 10, 209 gntnggcggt tttccagccc gttnatgcag ttnggttgtt gctnngaaca gcaagaatat 60cccccngaac aacataatca ggtcgngtcc ggagaaggag aaatcccatg acggtaaatn 120gcggtttggt cagngtgacc atccatgaaa tcagcgacag cagccccaga cgcataatca 180gcgccagtga taaccccagc aaacgcgctt tatcgngttg ttttggcggc agtttgtcag 240caagaatggc gatgaagacc aggttataga tacccagcac aatttcgaga acaaccagcg 300tgagtagccc cgcccaaatt gaggggtcca ttaagncnta cgaaant 347 10 339 DNAStreptococcus suis iri 11 10 ttcatcctng tcgagngggg aaatggggca cagttgttttccaattgata gaatttttaa 60 gaacctatat atanaaacat ggataggttg tttaatattttttnacacaa gatattgatt 120 tttgttttgt gaagtgctac actaatagaa gtgaagaattttggaggttt gtcagatgaa 180 tgtgcaagaa aatgtcttac cgagtataga attattggttttgaaacgtg atggacggac 240 agtatccttt gaccangata agattttttn tgctcttcagcgggcaaacc aagaattaga 300 acatcctgtt tcagnggcag gtttaaaaat tgtattaga 33911 270 DNA Streptococcus suis iri 13, 15, 27 11 ggaggnggtt anacnggcatttgcagatgc cgaatttatc taatgatttt gtagaagagt 60 ggtngcatng aacaaccctctttatcntta agaaaatgnt aggatagtcg gtcaatctan 120 gctatactag aaacgttattaagtcccgaa aaggtagttt atagactagt taatatttgc 180 agaaacactt gnaacacaattaaagaaact ggtantattg aatagtaagc gtaaaaactt 240 tactacactt cagtcactattttacntcaa 270 12 288 DNA Streptococcus suis iri 14 12 tattnntagtgtactataga aaaaactaac ntaccacaan acgtgatagg ttagttaant 60 taatgacattgggctntttg cccagcntct tttttnttat attagacagt atgtaggagg 120 tggntangttagaaaattgg ttaaacacca aacaaggtca ggtgtttcat tacaagatgg 180 aaaagattgagtatgcccta gaactgctag ggaatcccca gttngcagtt ccggtcattc 240 atgtcgctggaactaatggc aagggatcga ccattgcctt tatgcgca 288 13 342 DNA Streptococcussuis iri 16 13 gtacggcaac cggaagataa caagggatnt ggcngcagtt ggcagntcaaatcggcattc 60 cttagtantc tgtcaacttt gaaaaagagt actgggaccg cgtatttgagtactttttag 120 cagagtatcg ggntggtcgc acgcccnatc cagatgtcan gtgtaacaagganatcaagt 180 tcaaggcttt nttggantac gntatgaact tgggtgcgga ctatgtggcgacagggcact 240 acgctcaggt nacccgcgac gangacggca ccgttcatat gctgcgtggggcagataatg 300 gtaaggacca gacctatttc cncagccaac tctcacgnna ac 342 14 340DNA Streptococcus suis iri 18 14 ctgtctaacc accctaacat ngcatantcctcctttttta tctattttat caaaaaatcg 60 gngcttttct accatttgtc aagttcatcaaggtatttga cgaaaaatan tnngtgtctc 120 gtcatccaaa taaggaaatt gttttattttggactaaagt tacgtgtaaa aagngcatac 180 aaaaccaaca ccttntgttg naattttttgataaggtgtt acaatgatag agcataaaca 240 gttttaccga ttttgggtng aagcgtaatcgtnaaatttg ttatgcntaa tgaggtaata 300 cattgtccga atgagacgat gtatggaggcgatcgnangn 340 15 355 DNA Streptococcus suis iri 2 15 ancctttcnnggcccnatgg atgtttcgng gagaaaattg gaaaaataat cgggattatc 60 cagggaaatntcaaaatcac agttatccgt gaaacaagag caacngangt tgcnaagtaa 120 atgtagaagagtccgaaggg ctctttttnt actggctcaa agttcgttan gggttgggaa 180 tagaaaatagaaaatatttt aatcgtattt aaaagcagtt gaaattcatg ctaaattttg 240 ttacactagaatgaaagatt taaaaggaga tatcatgaaa gagcgaggct tactcatngt 300 cttttctggtccatctggtg cnggaaaagg aacagttcga aaggnaattt ntgaa 355 16 337 DNAStreptococcus suis iri 23 16 gntctngtag tagataagat tgaaacgcca aattcntcgtaacaaaacca agattgaacg 60 aaagcntcgt caaaaagtgg caactggtca agtctttacagatgaacttg ttgngcaaac 120 aggcgaggaa gtaaaagtgg ttcgtactaa gcaagtagacttgaaaccaa tggatatgga 180 agaagcagtc ctccaattgg agttgctcgg acatgatttctttatctata cagatgctaa 240 tgacggtaca acaaatgtat tgtatagacg cgaagatggagatttgggtc ttctagagnt 300 acgtcaataa agataataaa acagcncnan cgnannn 33717 350 DNA Streptococcus suis iri 24 17 agaagagaaa tgggggaacc tggnagttctacaagaaatt agttttgaag aacaggacgg 60 ggctagtcta tttgcgaaaa cgcaagagttcagcaaattc ttgctttttt tgatataatg 120 gtagaagcag ttttaagagg tatccaggtatgaatattca acaattacgc tacgttgtag 180 ccattgcaaa cagtggtaca tttcgagaggcggctgagaa aatgtangtg tcccagccta 240 gntngtccat ttccattcgt gatttggaaaaagagttagg ttttcaaatt tttngccgaa 300 ctagttcagg aacttttttg acacaaaaagggatggnact cacggagata 350 18 333 DNA Streptococcus suis iri 29 18gcgtatgggg gaaattgccc aagatgttan ccggtgacaa aatcacagat gcagcccgta 60atcangcaaa agaattagta gaaaaaggtt nggcgactag cctttttgct catgtccaaa 120gtttattttt tagttaaaat ttgttataat agatggcaaa attgaagaga attgtaggnt 180gaaatatgtc aaagattaag attgttacgg attcaagtac gactatcgaa cccagtttgg 240tcgaagaatt gaatataaca gttgttccnt tatctgtaat ggttgacgga gtcgtatact 300ctgacaacga tttaaaagaa ggcgaantcn tag 333 19 341 DNA Streptococcus suisiri 3 19 aggntgctan gaaaaaattg gctcacaaat catttctttt anttgacgattgcctttctt 60 tngatttngg tgatttactt tagtggaata gataaacgtt ggattattttggcaagtttt 120 cttnacttca ttccatcgca gattttatac cgtcgtcgcc taagagagcgactccaagaa 180 gaccagccca agnaggcngg ttttttgatg tgtaaattgg actacaattctttattaact 240 gtgctataat agtttttgca gaaaagtaaa gacggnggct ctaatttctgaaaggtaggt 300 ggtgtctatg ggcaaatcat cnaaatctna cagaaaggag n 341 20 264DNA Streptococcus suis iri 32 20 gaatcgaatt ggagntcgcc cctcaaacggctggcatatc ttttcaatcc ttatctntna 60 gtcgcaagcg acaagganta gggatnatataatctcctga gaatactgga ctcactgagt 120 ctggtatttt cattttatgc tataatggtttcatgacaaa tcgaatttta gatatggaac 180 aaatgcagga cgaggaatat gtcgngcgtaccctgcgtcc ncagaaatta aacgaataca 240 tcggtcagga caaggttaag gacc 264 21338 DNA Streptococcus suis iri 34 21 acagtagcct atgaantctt ggaagnaagcgggaagaagc aaaccattag tttcgaccaa 60 attttagttc ccataggagg aggtggtctggttgcaggcg tttcggccta tntgaaagaa 120 catgcacctg aaattangat tgttggtgttgaagcaagtg gggcacggtc aatgaaagcg 180 gctttngata aaggtcgtcc ggttnaattagaccaaattg ataaatttgc tgacggtatt 240 gcggtacaga aagtcggtaa gtcgacctacgaagtggctc ggaaatacgt agatcgcctg 300 attngtntgg atgaagggtg gatttccgggantatttt 338 22 351 DNA Streptococcus suis iri 4 22 aaaatggcaggggggaccca agggaantct tttctgatat caagggacaa cctggtcagt 60 cagntggntcaantacaagc cttaccactn gaacaaatan tcgaaaaccg ttatcaacgc 120 tttagaaaatantaggaaga cctagnattt ttttgataga tttgatacaa tggataaaat 180 aatttcaggaggttttccat gttagtaaaa gcagatctat caaacgcagn agaattgcta 240 cntattcagcnccgagcatt tgcggcttta tataaaacct atcaggacca gtacaaccct 300 gccattgnaactatggacta tttccaatca cgctttgcac gaccaaattg t 351 23 362 DNAStreptococcus suis iri 7 23 gaaattgatg ggcatctttg gtattaatag gaactccatggctcaatctt cttcggttta 60 ttggtaatag tagttaccgt cangaanaaa tcgcaaagtataaaaagtgc tgtgaagaga 120 aaaaaagaaa ataagaatct ttctaaacaa gataagagccgtcaggctct tttttngata 180 taatatagtg gatatggtta attaaaattg tcagaaaagactattttana gattaacact 240 ctctgaaaat cntcattaac aagaaaagag gcngggctcaagccccgcat cacntctcaa 300 agttagcgtc aacatctcag cgcagtagtg gtngattgggtttaacagtc cagtggagtg 360 tc 362 24 362 DNA Streptococcus suis iri 8, 2624 ttcggaatcc ttctntctcc attggaacag ggatacaaag ggacgttaag gaaatccgta 60ngaaaatagg aaattgacgc agtgtgctan acacacaggg aagtttatct ttttccacta 120ggattttagt ccgtgttcaa ctaagatacg agatatgttc tggtttacca gaaatttcng 180nagaaaatta ggagactgac gctgagtgtt aacactcaag gaaggctatc tatttttcta 240agaaattaat ctcgagttca atttcttntg attagtaaat aaatgaattg tatctatttt 300ttggggtatc gccaagcggt aaggcaaggg actttgactc cctcatgcgc cggttngcat 360 cc362 25 405 DNA Streptococcus suis ivs 1 25 aatgatgttt gataaacacgccaatctcaa atacaaattt ggtaatcgtc atttctgggc 60 agagggatat tatgtaagtacggttggact aaatgaagcc acaattaaga aatatataca 120 agaacaggaa aaacatgatatagcacttga taagttgagt gtaaaagagt atgaagatcc 180 ctttagggat aatggcaagtagtacgaatg cctctttaag aggctagtga cgagtcaaaa 240 gcagtgaggc ttgaacaaagtgaaagccag cgtctttagg cgctggctgg tgatgtgggc 300 ttatagccct tgttcaaaccacccgtttga cgggtggtca tgattttttt tgaatatttt 360 tcactatttt gttttacaaactagccacct tgtgttagac tatag 405 26 410 DNA Streptococcus suis ivs 11 26taccaccata tcaccaatat cacgcgccca gatgcgccaa tcgaagtggt ggatgtggct 60ggttcccttt gtgaaaacaa cgacaagttt gcggtcaatc gtgaattacc acgggtagaa 120gtaggagaca ccttggtcat tcatgacagt ggggcccacg gcttctccat gggctacaac 180tacaacggtc gtctgcgttc ttctgaaatc cttttgcagg aagatggcac agcgcggatg 240attcgtcgtg ctgaaacacc agaagactat ttcgcaacta tttacggttt tgattttgac 300aggtaagtct tggaaaagac tagggaattt ggtataatag ggttattgaa agattgttaa 360aaacaatcag aagtatactt tttagaagag tcaggagatt gacagatgaa 410 27 412 DNAStreptococcus suis ivs 15 27 gggctatggt ataattaaaa gacatgtata gtcagaatgaaaatgaattg attgccattg 60 gtgagagaat tggaaaggcc tgtaagccaa atcaagttctagtattatca ggggatttgg 120 gtgctgggaa aacaactctg accaagggtt tggccaaggggttaaaaatt gaacagatga 180 ttaagagtcc tacttatacg attgttcgag agtatgagggggccatgccg ctctatcact 240 tagatgttta tcgaattgga gatgaccctg actcgattgatttggatgat tttctctatg 300 gaggaggtct aacggttatc gagtggggag aattactggatgtcagtcta tttgatgact 360 atttgctcat tcgtatagag aaagagggag atggtcgacgattgacagtc ga 412 28 449 DNA Streptococcus suis ivs 16 28 gaaaattgttgttgttttgg aacactagta gaccagaggc ttctagtaag gtagttgtgc 60 tcactgaggagggggaagga tgatggaagt tgagaaaagg agtaaggatt atgctcgtat 120 gtttgaccagcaagtcggtc tttatgaaga ctatgctcgt ggacatggac tcaatgcaaa 180 atgtttatccattctcatgt ggatttatta taatcccgga ggtgtgacgc aaaactgggt 240 cagtaagaagacctattcaa gcaaacaagt tgtcaatgct actgtaaaga aatttttgga 300 tggaggcctggtagttctag aggagaatcc agcagataag cgacataaga aaattaaatt 360 gacagaggaggggcaacaat ttgctagtcg cattttggat cccttagagg aggcggaaaa 420 taaggcgctgtctcaactca gtcaggagg 449 29 410 DNA Streptococcus suis ivs 18 29gcgttttgga acaagtacgt taagagaaac ctagagaaat ctagggtttt tgcttttata 60tatctttaca ttgtttaaag aaaatagcat ttcaaaaact ttttgaaaaa aatgtgatat 120tctgagcata ttttttgaaa tcggtaacat ttatattgta taatatagtt cgtaaaaaaa 180tatattttcg aaagtgagat tttacattat ggctaaaatc gttgttgtcg gtgctaacca 240tgctggtact gccgcaatca aaactatgtt gacaaattat ggtcaagaaa atgaaatcgt 300tgtatttgac caaaactcac atatttcatt cttgggttgt ggtatggctt tgtggatcgg 360tgagcaaatt ggcggtcctg aaggactctt ctactcaaac aaagaagagt 410 30 437 DNAStreptococcus suis ivs 19 30 tcgttccatt tgctggtgaa atgcccagca atacgcttcntagcaataga agaaccaaat 60 agatggcact caatttcatg aggaagaaca gaagagtaaaaagcctgtct aaccacccta 120 acatagnata ttcctccttt ttcatctatt ttatcaaaaaatcggtgctt ttctaccatt 180 tgtcaagttc atcaaggtat ttgacgaaaa atattttgtgtctcgtcatc caaataagga 240 aattgtttta ttttggacta aagttacgtg taaaaagtgcatacaaaacc aacaccttat 300 gttgaaattt tttgataagg tgttacaatg atagagcataaacagtttta ccgattttgg 360 gttgaagcgt aatcgtaaaa tttgttatgc ataatgaggtaatacattgt ccgaatgaga 420 cgatgtatgg aggcaat 437 31 417 DNAStreptococcus suis ivs 2, 4, 28 31 aagacggcgt caaggatgac aatcttgtggtgacgaccac ccagaaactg gcgtagcntt 60 taccgtggcc ggaatcatga tcgcggttttccagccgttc atgcagttcg gttgttgctt 120 tgaacagcaa gaatatcccc ccgaacaacataatcaggtc gcgtccggag aaggagaaat 180 ccatgacggt aaaatagcgg tttggtcagcgtgaccatcc atgaaatcag cgacagcagc 240 cccagacgca taatcagcgc cagtgataaccccagcaaac gcgctttatc gcgttgtttt 300 ggcggcagtt tgtcagcaag aatggcgatgaagaccaggt tatcgatacc cagcacaatt 360 tcgagaacaa caagcgtgag tagccccgcccaaattgagg ggtccattaa gaattcc 417 32 444 DNA Streptococcus suis ivs 2032 agttcagatg ttcggtttag gaattgccgg cgtctggctg tcgattttga tggacctgct 60cttgcgagcg atttttctga cttggaggtt tattgtgcaa acacgaaaac tggctgaata 120ggctagtttt ttggtataat atcagtagaa tgataaaaag gagataatca gatgaaaacc 180attcacacag ataaggcacc tgcagcaatt ggcccatacg ttcaagggaa ggttgttgga 240aatttcctat ttgcctctgg tcaagttcct ttgtcacctg aaactggtga agtggttggt 300gaaaccattc aggagcagac tgagcaagtc ttgaaaaata tcgcagcaat tttatcagaa 360gcaggaacag actttgacca tgtggtgaag acgacttgtt tcctaaaaga tatgaatgat 420tttgtagcct ttaatgaagt ttat 444 33 480 DNA Streptococcus suis ivs 23, 2433 tctgcactgt tgcgctgcct ataagttcta cgttcagtag tagatgaaat gttcagagga 60agtggtatgg gttccaactt agtaaaatta gtcattgatg atttggcgaa cagaaattcc 120aaagcctttc aaatcgcagt tgaagaagag aaattgggaa cctggaagtt ctacaagaaa 180ttagttttga agaacaggac gggctagtct atttgcgaaa acgcaagagt tcagcaaatt 240cttgcttttt ttgatataat ggtagaagca gttttaagag gtatcaggta tgaatattca 300acaattacgc tacgttgtag ccattgcaaa cagtggtaca tttcgagagg cggctgagaa 360aatgtatgtg tcccagccta gtttgtccat ttccattcgt gatttggaaa aagagttagg 420ttttcaaatt tttagccgaa ctagttcagg aacttttttg acacaaaaag ggatggaatt 480 34418 DNA Streptococcus suis ivs 25 34 ggagatagca atgcttaata tttttgtattagaagatgat ttttttcagc agagcaggtt 60 agaaaatgct attaggcagt gtgttgaagaaacgtcagta aggtataaat tcctagaagt 120 ttttggtaaa ccaaatcaat tattggaatcaattgaggaa gcagggaatc atcaattttt 180 ctttttagat attgaaataa aaggagaagaaaagaaagga atggaaatcg ctaaagaaat 240 ccgggctcga gatccttatg ctgctattgtctttgtaaca actcactcag aatttatgcc 300 agtaacatat cgttatcagg tttctgctttagattttata gataaaggcc tggaggatng 360 tgactttcaa aaggcagtat cagatgtcttagtgcatgct tttgaaaata ttgatcat 418 35 446 DNA Streptococcus suis ivs 2935 ggcaagggtg ggtaaatttc taattggtga caaggcactt gaattctacc cagatagcaa 60cgttgaacgc tatatccaga ttccttggtc agaaatgact agcattggcg caaaacgttt 120ctggcaaagc aatcagccgt cattttgaaa tttatacaga gaaaagtcga tttcttgttg 180gcatctaaag attctggtaa gattcttaaa attgcccgtg agcatatcgg caatgaaaaa 240gttgtgaaat taccgactct tatgcaaaca atcggcagaa aaatttcgaa tctatttgcc 300aaaaaataaa aattcaagta taatagtaga aacggataag tagcatctgg ctccttccag 360aaagtctgcg gtcgctgtga gcagatagga aaaagttgtg aaattctacc gttatgaaat 420tatcaaaata caatcaagtg cacaga 446 36 416 DNA Streptococcus suis ivs 3 36ggattatcta ctataagcag tattcagaag ggcatgagga caagaaatcc tacaagattc 60tacaagaagt aggcatgagc cagaaggctg tcaagaaaac aattaactcc caaacactta 120cggtcttctt tatgcctttg gtcatggcga ccctacactt tgtcatcgcc cttatcatgc 180tcaagcaaat gctactaagt tttggtgtta cctcatcact aatgatttac acagtcagtg 240gcatcaccct actggcagtc actctgattt actttgtcat ttacaagtgg actagtcgca 300cttattatcg cattattgaa cggtagcaga agtctcgcct tgtgcgagat ttcttgcttt 360ttcagggaaa tggtgttaca atggtaatac caaaggaata ctcgaagagg tgagaa 416 37 263DNA Streptococcus suis ivs 31 37 acgaaaatdg atggatccat gcataaactgcatcccttaa cttgtttttc gtgtgcctat 60 tttttgtgaa tcgaattcga gctcgcccctcctgaccacc tatntgcatc aagtgccaaa 120 tgaccagtcg agtgtgcggt tagacaactactatacgggc aaggaactgg agattgagtt 180 ggatgtggct ttgactccta gccaaaatgcccagcggtac ttcaagaagt accagaaact 240 caaggaggcg gtcaagcacc tga 263 38403 DNA Streptococcus suis ivs 32, 35 38 atatttgctc tcctgctctttaggggacaa tggaaaaagt agtctgtatc caacatttta 60 caaagtagga ttttttctataaaatagatt gtatatgaca ttcaaatcca ttctcaaaca 120 actcaaacta tttgattatatcttaatcgg attcacccta gttttatcct ttcttccagc 180 aatttttacc tacacacaactgacaacaga tgcaaatgag gcaaaaacaa ttgcctatgt 240 ccgcatcaat ggtgaggtggtcgaccaatt tgaattatca aaggacacac cccgtcaaga 300 aaagacctac tatcccaatgaagggcaata caatatcatt gaagttgatg gcgaacgcat 360 tcgtgtcaag gaagacaatagcccagacca aatcgccgtt atg 403 39 401 DNA Streptococcus suis ivs 33 39actcagttga acggagtagg atttataggt aaattgcctc caaatatcgt aagacaatcc 60tctattgaaa aataggggat tgtttgttta gaaataatgg tggagattct gtaaaaagcg 120aaagtggttg gaaagttagg gtttagccga gaaaaagaga cttttctatc tatctttcac 180aattttctgt caatttgtgg tagaatagaa aaaatagatt ttttatgagg gataccatga 240cattagtata tcaatcaaca cgcgatgcta aaaatactgt atcggctagt caagcgattt 300tgcagggctt ggcgaccgac ggtggtttgt ttacaccgct ttctattcca acagttgact 360tggatttttc tgttttgaaa gatgcttctt atcaagacgt t 401 40 404 DNAStreptococcus suis ivs 34 40 gtttatcgtt cgctggagga aaagggctat aatccgattaaccaaatcat tggctatgta 60 ttaagtgggg accctgctta tattcctcgc tataatgatgcccgcaatca gattcgtaag 120 catgaacgag atgaaatcat tgaagaattg gtgcgctactatttgaaagg gaatgggatt 180 gacctctaat gagaataatg ggattagacg tcggttccaagacagttggt gtagccattt 240 cagatccgtt aggtttcacg gcccaagggt tggaaatcatcccaatcgat gaagaaaagg 300 gcgaattcgg tctggagcgt ttgaccgaac ttgtagaacagtacaaggtt gataaatttg 360 ttgtaggctt gccgaagaat atgaataata ctagtggtccacgt 404 41 384 DNA Streptococcus suis ivs 36 41 ggtataatta tctgataaaaaactttggag acgacagtga gtttagaaaa ttacatgccg 60 gattttgcct tggaaaaggcttatgacgtg accgtcgaaa gcttgaaaaa acatggcata 120 aaagtagtgt ttgttgacttggataatacc ttgattgctt ggaataatcc cgatggtacg 180 ccagagatgc gccagtggttacatgatttg caggacgcag gtattcctgt tgtggtggtg 240 tctaacaata aatacgaacgtgtcaaacgg gcggttgaaa aatttgggat tgaatttgaa 300 gccttcgctc tcaagcctttcacctttggg attaaccgtg ctttgaaacg ctttgatgtc 360 cagccgtatg aggtaattatgatt 384 42 413 DNA Streptococcus suis ivs 5, 10, 12, 22 42 acgcacttgctcgcgtagtc gatgaattag atgtacccgt tatggctttc ggtcttaaaa 60 atgatttccgaaatgaacta tttgaaggtt cccaacattt gctcttattg gctgataaat 120 tagatgaaatcaaaacaatc tgccaatatt gttctaaaaa agcgacaatg gttttgagaa 180 cacaggatggaaaacctact tatgaaggag aacaaatcca aattggtggc aatgaaacct 240 acattcctgtctgtcgcaaa cattattttt caccagaaat taaagattta ccctaatttt 300 tgaaaatgaaatgagaagca actgtaaact gagcaactat atagaactga atttgcctat 360 gactctgtgccaattttcat aacttacata ctacggcaaa ggaattgaac acg 413 43 428 DNAStreptococcus suis ivs 6, 7, 13, 14 43 gaagggatta aacaatccta tgctattcaggctgttcgtg aaattcggat tatcgttcat 60 cctaacaagg tcactgatga tcagattaccatcttggccc atgatgttcg tgagaaaatt 120 gaaaataatc tggattatcc aggaaatatcaaaatcacag ttatccgtga aacaagagca 180 acagatgttg ctaagtaaat gtagaagagtccgaagggct ctttttctac tggctcaaag 240 ttcgttttgg gttgggaata gaaaatagaaaatattttaa tcgtatttaa aagcagttga 300 aattcatgct aaattttgtt acactagaatgaaagattta aaaggagata tcatgaaaga 360 gcgaggctta ctcattgtct tttctggtccatctggtgcc ggaaaaggaa cagttcgaaa 420 ggaaattt 428 44 383 DNAStreptococcus suis ivs 8 44 cttcaaagga ccccaggacc tttgaattct caaatacgcatcatgttgac agttgccaca 60 cctacaccaa aatcaaatgc caacaagcgt tgagtcgggtaatagcgtaa gtagcgcaag 120 gtcatgataa gctgctcttc catacttaga cggcgtgggcgtcctccttt tcggtgttgc 180 tcttgataag cgtcagtgag acaatcaagc atcagatgaaacgtcgcttt tttacaccta 240 tcaacaattt gaaattctct gagtttaatt ttaagactttttcgtatgtt gtttccatac 300 ctttagtata ccgcctttga gttaccgaac aagtctattgctaaacttga tgaaggttgt 360 attgtctgtt ataatattgg ata 383 45 415 DNAStreptococcus suis ivs 9, 17 45 gcctatgaga ctcattttcc ctgtctcaactgctctaagc aattgttaca ggttggttgt 60 aagcgggttg tctatatcaa tgaataccgcatggatgact atgctcagta cttgtataaa 120 gaaaagggct gtgagttggt tcatttgcctctagaggtgg ttaaacaggc atttgcagat 180 gccgaattta tctaatgatt ttgtagaagagtggttgcat agaacaaccc tctttatctt 240 taagaaaatg ctaggatagt cggtcaatctatgctatact agaaacgtta ttaagtcccg 300 aaaaggtagt ttatagacta gttaatatttgcagaaacac ttgaaacaca attaaagaaa 360 ctggtaatat tgaatagtaa gcgtaaaaactttactacac ttcagtcact atttt 415 46 45 DNA artificial sequence PCR primercorresponding to positions 250 to 273 of the fbps gene 46 gcggatccgatgacgatgac aaatcttttg acggattttt tttac 45 47 32 DNA artificial sequencePCR primer corresponding to positions1911 to 1892 of the fbps gene 47cccaagcttg ggcatgaact agattttcat gg 32

What is claimed is:
 1. A process for modulating virulence of aStreptococcus comprising: modifying a genomic fragment of theStreptococcus; wherein at least part of the genomic fragment is capableof hydridizing to a nucleotide sequence selected from the group ofnucleotide sequences consisting of any one of SEQ ID NOS: 8-45 orfragments thereof; and generating a clone having the modified genomicfragment.
 2. The process according to claim 2, wherein the genomicfragment comprises a functional part of a gene, the expression of whichcan be environmentally regulated by iron-restricted conditions inStreptococcus suis.
 3. The process according to claim 1 or 2, whereinthe genomic fragment comprises a functional part of a wild-typeStreptococcus suis gene expressed in a pig infected with wild-typeStreptococcus suis.
 4. The process according to claim 3, wherein thewild-type Streptococcus suis gene encodes afibronectin/fibrinogen-binding protein.
 5. The process according to anyone of claims 1 to 4, wherein the Streptococcus is Streptococcus suis.6. The process according to any one of claims 1 to 5, wherein modifyingthe genomic fragment comprises functionally deleting the at least partof the genomic fragment capable of hydridizing to the nucleotidesequence.
 7. A clone of a Streptococcus, obtained by the processaccording to any one of claims 1 to
 6. 8. The process according to claim1, wherein the genomic fragment encodes a fibronectin/fibrinogen-bindingprotein.
 9. A process for assaying virulence of a Streptococcuscomprising: assaying an ability of the Streptococcus to infect asubject; wherein the Streptococcus comprises a genomic fragmentassociated with a virulence factor to infect a subject; and wherein atleast part of the genomic fragment is capable of hydridizing to anucleotide sequence selected from the group of nucleotide sequencesconsisting of any one of SEQ ID NOS: 8-45 or fragments thereof.
 10. Theprocess according to claim 9, wherein the genomic fragment encodes afibronectin/fibrinogen-binding protein.
 11. An isolated or recombinantnucleic acid molecule of a Streptococcus origin comprising a nucleotidesequence capable of hybridizing to a nucleotide sequence selected fromthe group of nucleotide sequences consisting of any one of SEQ ID NOS:8-45 or fragments thereof.
 12. A vector comprising the isolated orrecombinant nucleic acid molecule of claim
 11. 13. A host cellcomprising the isolated or recombinant nucleic acid molecule of claim 11or the vector of claim
 12. 14. The host cell of claim 13, wherein thehost cell is of a Streptococcus origin.
 15. A vaccine comprising theclone of claim 7, the isolated or recombinant nucleic acid molecule ofclaim 11, the vector of claim 12 or the host cell of claim 13 or
 14. 16.A protein or fragment thereof, encoded by the isolated or recombinantnucleic acid molecule of claim
 11. 17. An antibody directed against theprotein or fragment thereof of claim
 16. 18. An antigen comprising theprotein or fragment thereof of claim
 16. 19. A diagnostic testcomprising the antibody of claim
 17. 20. A vaccine or diagnostic testcomprising the antigen of claim 18.